An improved method for isolation of active vitellogenin messenger RNA from chicken liver. Use of diethylpyrocarbonate

338

Biochimica et Biophysica Acta, 517 (1978) 338--348

© Elsevier/North-Holland Biomedical Press

BBA 99094 AN IMPROVED METHOD F O R ISOLATION OF ACTIVE V I T E L L O G E N I N MESSENGER R N A F R O M CHICKEN L I V E R USE OF D I E T H Y L P Y R O C A R B O N A T E

JEAN-PIERRE JOST, GUNDULA PEHLING, SAKOL PANYIM and TAKESHI OHNO With the technical assistance of MONIQUE SELDRAN Friedrich Miescher-Institut, CH-4002 Basel (Switzerland)

(Received August 9th, 1977) Summary 1. In chicken liver homogenate containing 15 mg protein and 1.2 mg R N A per ml, diethylpyrocarbonate (60 mM) at pH 6.5 and r o o m temperature inactivated liver ribonucleases by over 90% whereas heparin (4 mg/ml) and yeast R N A (6 mg/ml) under similar conditions inhibited ribonuclease activity by 30--40%. 2. Maximal recovery in polysomes from homogenate containing 60 mM diethylpyrocarbonate required the presence of exogenous RNA. 3. The recovery in total m R N A and vitellogenin m R N A activity from liver homogenate can be improved by 3- and 7-fold, respectively, by adding 60 mM of diethylpyrocarbonate to the homogenization buffer containing already heparin and yeast R N A as inhibitors of ribonucleases. 4. Vitellogenin m R N A purified from homogenate containing diethylpyrocarbonate had a molecular weight of a b o u t 2.4 • 106 and coded for a major peptide of molecular weight 220 000 in a reticulocyte cell-free system.

Introduction One major difficulty in isolating non degraded polysomes and m R N A from eukaryotic tissues is the presence of large quantities of ribonucleases in such tissues. This difficulty has been partially overcome b y adding various inhibitots of ribonucleases to the cell or tissue extract. Among those most c o m m o n l y used are: soluble yeast R N A [1--4], heparin 1,3--6], polyvinyl sulfate [7], bentonite [8,9], transition state substrate analogues of ribonucleases [10,11] and rat liver supernatant fractions [12,13]. These inhibitors only mask the ribonuclease activity w i t h o u t removing or destroying it. An alternative possibility is to inactivate the ribonucleases by reaction with diethylpyrocarbonate

339 [14]. Recently, Ehrenberg et al. [14] have extensively reviewed the potential use of diethylpyrocarbonate in the isolation of nucleic acids. In all experiments to date where diethylpyrocarbonate has been used in the isolation of polysomes it resulted in full size b u t less active or inactive polysomes or m R N A [5, 15]. Since the rate constant for the reaction of proteins and nucleic acid with diethylpyrocarbonate differs by several orders of magnitude in favour of the proteins [14], it appears likely that conditions exist where the nucleases will be destroyed b u t where the polysomes and m R N A are n o t inactivated. As a test of this hypothesis we have studied the effect of diethylpyrocarbonate on the isolation and purification of vitellogenin m R N A from the liver of estradioltreated chicks. Materials and Methods

Animals and hormone treatment For all experiments white Leghorn male or female chicks weighing 200--300 g were used after a 20 h fast. Chicks were injected intramuscularly with 50 mg per kg body weight of 17~-estradiol (dissolved in propylene glycol). The animals were used 31~ to 4 days after initial injection of 17~-estradiol.

Chemicals 17~-Estradiol and heparin were obtained from Ciba-Geigy Limited, Basel. Diethylpyrocarbonate and yeast R N A (Torula species) were purchased from Serva Feinbiochemica (Heidelberg, G.F.R.) Ribonuclease-free sucrose from Schwarz Mann was used throughout all experiments. Oligo(dT) (T3) was purchased from Collaborative Research Inc., Waltham, Mass., U.S.A. L-4,5-3H] leucine 56 Ci/mmol was purchased from Amersham, Radiochemical Centre.

Preparation of polysomes Livers were perfused with 50 ml of ice-cold 0.2 M ammonium acetate, pH 6.5, containing 0.01 M MgC12 and then frozen at --20°C in 50% glycerol, 0.2 M ammonium acetate buffer, pH 6.5. Frozen livers (15--20 g) were .minced in 10 volumes of 0.2 M ammonium acetate pH 6.5 containing 10% glycerol, 5% Triton X-100, 0.01 M MgC12 plus the indicated inhibitors of ribonuclease at --2°C. In our final procedure we used 6 mg yeast RNA, 4 mg heparin and 10 pl of diethylpyrocarbonate per ml of buffer. Minced liver was homogenized with three strokes at 250 rev./min in a loose fitted glass teflon homogenizer. The homogenate was centrifuged immediately at 25 000 × g for 7 min in an angle rotor (SS.34 Sorvall) at -2°C to sediment chromatin and cell debris. The supernatant fluid was filtered through 4 layers of sterile cheese cloth and the clear solution was overlayed on 8 ml of 40% sucrose containing 0.2 M ammonium acetate pH 6.5, 10 mM MgCI~ with 2 mg heparin/ml and centrifuged at 105 000 × g for 70 min at 0°C in a Beckman 30 angle rotor.

Deproteinisation of RNA The clear polysome pellet obtained from 15 g of liver was resuspended by gentle homogenization in 30 ml of 0.05 M sodium acetate, pH 5, containing 0.01 M EDTA, 2% recrystallized sodium dodecyl sulfate. After addition of 30

340 ml of redistilled phenol containing 0.1% hydroxyquinoline, the polysome fraction was extracted for 3 min at room temperature. 30 ml of chloroform was then added and the extraction continued for 3 min. The phases were separated by centrifugation and the water phase and interphase were re-extracted two more times as described above. The water phase and interphase were then extracted 3 times with 60 ml chloroform and the R N A from the water phase precipitated with 2.5 volumes of cold ethanol and 0.2 M sodium chloride (final concentration). After standing at 20°C for at least 3 h, R N A was collected by centrifugation, washed once with cold 3 M sodium acetate pH 5.5, 0.001 M EDTA and once with 70% ethanol, 0.2 M NaC1 (--20°C). The RNA pellet was either used immediately or frozen in liquid nitrogen.

Oligo(dT)-cellulose affinity chromatography Separation of m R N A from total polysomal RNA was carried o u t as outlined by Bantle et al. [16].

Lithium dodecyl sulfate-sucrose density gradients Approximately 10 A260 units of total m R N A {post oligo(dT)-cellulose fraction) dissolved in 100 pl of 0.01 M Tris, pH 7.0, 0.001 M EDTA, 1% lithium dodecyl sulfate, and was heated for 5 min at 68--70°C. After rapid cooling samples were loaded onto linear 5--17.5% sucrose gradients containing 0.02 M lithium chloride, pH 6, 0.005 M EDTA, 0.25% sodium dodecylsulfate. After 6 h of centrifugation at 40 000 rev./min in a SW 40 Beckman rotor at 6°C, gradients were fractionated and the absorbance of the effluent was monitored at 260 nm with a Zeiss PMQ II recording spectrophotometer. After overnight precipitation with 2.5 volumes of cold ethanol, 0.2 M NaC1 a t - - 2 0 ° C , R N A was recovered by centrifugation at 105 000 X g for 60 min.

2.3%.polyacrylamide-sodium dodecyl sulfate gel electrophoresis RNA samples were analyzed by electrophoresis on 2.3% polyacrylamide gels for 6 h at 5 mA per gel as described by Loening [17]. Prior to electrophoresis RNA was denatured at room temperature in 90% deionized formamide pH 7 and directly loaded onto the gels. After electrophoresis, gels were soaked overnight in water and analyzed at 260 nm in a Zeiss PMQ II recording spectrophotometer.

In vitro protein synthesis and radioimmunoprecipitation The rabbit reticulocyte lysate system was prepared as outlined by A b e t al. [3]. RNA fractions (0.1--10 pg/100 pl ihcubation mixture) were translated for 3 0 - 4 0 min in the lysate system as described by Palmiter [18]. At the end of the incubation aliquots of the incubation mixture were precipitated with 5% trichloroacetic acid and the " h o t trichloroacetic" precipitable counts determined. The rest of the incubation mixture was immunoprecipitated with antilipovitellin as previously described [19]. Immunoprecipitates were washed 3 times by centrifugation through a cushion of 0.3 ml of 0.9 M sucrose, 0.15 M NaC1, 1% Triton X-100, 1% sodium desoxycholate, 0.1 M leucine at 10 000 X g for 15 m i n in 2 ml plastic conical tubes into a HB-4 Sorvall rotor. Purified antigen-antibody precipitates were either dissolved in formic acid and directly

341 counted for radioactivity or were solubilized by boiling for 5 min in 50 ~l of a solution of sodium dodecyl sulfate-dithiothreitol as described by Palmiter et al. [20]. Upon separation of the proteins by electrophoresis on 5% polyacrylamide-sodium dodecyl sulfate gels [21], gels were stained and counted for radioactivity as previously described [19]. The wheat germ cell-free system was prepared as described by Roberts and Paterson [22].

Labeling o f R N A and purification o f yeast R N A t R N A was prepared from chicken liver according to M~ienp~i~i and Bernfield [23] and was labeled at the 3' end with tritiated potassium borohydride as described by Leppla et al. [24]. A specific activity of 300 000 d p m / p g R N A was obtained. Total liver RNA was uniformly labeled in vivo for 1 h after intraperitoneal injection of 1 mCi of [3H]uridine/100 g. Total RNA was extracted by the chloroform-phenol procedure. A specific activity of 500 d p m / p g R N A was obtained. Commerical yeast R N A was dissolved in 0.05 M EDTA and treated with 0.05% diethylpyrocarbonate at room temperature for 15 min. RNA was extracted 3 times with phenol at 65°C. Upon precipitation with ethanol, R N A was dissolved in water and extensively dialyzed against water. Upon lyophilization RNA was kept at --20°C. Ribonuclease assay 100-pl aliquots of supematant fractions obtained from the 10% (v/w) liver homogenate were incubated in triplicate with either 1 pg of [3H]tRNA or 100 pg of uniformly labeled liver RNA for 30 min at 35°C. The reaction was stopped with 2 ml of cold 10% trichloroacetic acid and after standing 1 h in ice, the precipitate was sedimented by centrifugation. Both supernatant and sediment were counted for radioactivity. A parallel control consisting of labeled R N A only was also incubated in 100 gl of the same buffer used for the homogenate. Results

Effect o f pH, yeast RNA, heparin and diethylpyrocarbonate on liver ribonuclease activity Ribonucleases in the liver are active over a wide range of pH values. As a first attempt to limit the action of ribonucleases we tested the pH-dependence of ribonuclease in crude chicken liver extracts. The inset of Fig. 1 shows that virtually 100% of [3H]tRNA was hydrolysed at pH 8, whereas at pH 6--6.5 a b o u t 60% of the substrate was cleaved. We then established a dose-dependent inhibition curve of liver ribonucleases for each of the inhibitors at pH 6.5. Fig. 1 shows that under our experimental conditions both heparin and yeast RNA inhibited ribonuclease activity by a b o u t 30%, whereas diethylpyrocarbonate, at a concentration of 60 mM (1%), inactivated ribonucleases b y over 90%. When uniformly labeled liver RNA was used as a substrate, essentially the same results were obtained as with [3H]tRNA. Diethylpyrocarbonate had very little effect on total liver nuclease activity up to a concentration of 30 mM (0.5%), then there is an abrupt transition that resulted in maximum inhibition a t 60

342 fO0

80

60

pit

'v 6 I

o

7 I

,

8

\

~oo

40 -

1D

¢-

e

Z n..

--

80

"to

20

~

< --

Z

--

--

600~'

J 1 heparln

J

2 o r yea,at

RNA

J

i

3

4

6

mg//ml

i

l-

15

30

I

I "

m M dlothyl p y r o c e r b o n a t e

45

60

Fig. 1. D o s e - d e p e n d e n c e c u r v e o f t h e i n h i b i t i o n o f liver r i b o n u c l e a s e s f r o m e s t r a d i o l - t r e a t e d c h i c k s b y d i e t h y l p y r o c a r b o n a t e ( c u r v e C), y e a s t R N A ( c u r v e B) a n d h e p a r i n ( c u r v e A). R i b o n u c l e a s e a c t i v i t y f r o m c r u d e e x t r a c t w a s t e s t e d as o u t l i n e d in M e t h o d s . T h e i n s e t r e p r e s e n t s t h e p H d e p e n d e n c e of r i b o n u clease a c t i v i t y f r o m c r u d e e x t r a c t s p r e p a r e d f r o m c h i c k e n liver. E a c h p o i n t r e p r e s e n t s t h e a v e r a g e o f t h r e e determinations.

mM (1%}. It is noteworthy that the concentration of protein and RNA in liver homogenate was 15 and 1.2 mg/ml respectively.

Effect of diethylpyrocarbonate, yeast RNA and heparin on the recovery of polysomes, activity of polysomal mRNA and vitellogenin mRNA We tested the effect of diethylpyrocarbonate, heparin and yeast RNA at their optimal concentrations on the recovery of polysomes (measured as polysomal RNA) and the activity of total and vitellogenin mRNA. The results in Table I show that yeast RNA and hepbxin separately markedly increased the recovery in polysomes as compared with the non-treated homogenate while also increasing the specific activity of viteUogenin mRNA. A combination of the two did not increase the recovery of polysomes but did have an additive effect on both total mRNA and vitellogenin mRNA-specific activity. The ~ecovery in total mRNA and vitellogenin mRNA activity from liver homogenate could be improved by 3- and 7-fold, respectively, by adding 60 mM of diethylpyrocarbonate to the homogenization buffer containing already heparin and yeast RNA as inhibitor of ribonucleases. Diethylpyrocarbonate alone gave very poor yield in polysomes. However, addition of yeast RNA to the homogenate

None Yeast R N A Heparin Diethylpyrocarbonate Diethylpyrocarbormte, RNA RNA, heparin Diethylpyrocarbonate, RNA and heparin

Additions

6 mg/ml 4 mg/ml 6 0 m M (1%) 60 raM, 6 m g / m l 6 mg/ml, 4 rng/ml 6 0 raM, 6 m g / m l , 4 mg/ml

Concentrations

1 30 27 8 30 30 31

Polysomal RNA ( A 2 6 0 n m / g Liver)

9 10 10 12 21 22 60

160 345 138 540 767 286 731

I+ 2 0 0 + 1034 -+ 5 1 2 -+ 8 7 5 _+ 1 0 8 5 -+ 2 6 6 4 -+ 6 6 7

Total proteins

100 438 555 715 1516 1146 8532

-+ 2 -+ 5 2 + 65 +- 4 2 -+ 2 1 0 -+ 1 5 2 -+ 4 2 5

ViteLiogenin

dprn//~g R N A i n c o r p o r a t e d i n t o

1 4.2 5.4 5.7 6.9 5.4 14

% of total

P o l y s o m e s w e r e p r e p a r e d f r o m 2 g o f liver in t h e p r e s e n c e o r a b s e n c e o f i n h i b i t o r s o f r i b o n u c l e a s e as d e s c r i b e d in M e t h o d s . U p o n e x t r a c t i o n of R N A w i t h c h l o r o f o r m - P h e n o l , p o l y ( A ) - c o n t a i n i n g m R N A w a s i s o l a t e d o n o l i g o ( d T ) - c e l l u l o s e a n d a s s a y e d f o r t o t a l p r o t e i n a n d viteLiogenin s y n t h e s i s in a w h e a t g e r m s y s t e m as indic a t e d in M e t h o d s . E a c h v a l u e r e p r e s e n t s t h e m e a n -+ S.E.M. o f t h r e e m e a s u r e m e n t s m i n u s t h e r a d i o a c t i v i t y o f t h e p r e c i p i t a t e o f t h e c o n t r o l i n c u b a t e d w i t h o u t e x o genous m R N A .

E F F E C T OF V A R I O U S I N H I B I T O R S OF R I B O N U C L E A S E ON T H E R E C O V E R Y OF P O L Y S O M E S , A C T I V I T Y OF T O T A L rnRNA A N D V I T E L L O G E N I N mRNA

TABLE I

O~ O~

344

containing diethylpyrocarbonate greatly improved the recovery of polysomes. Direct treatment of m R N A with as little as 3 mM diethylpyrocarbonate at room temperature resulted in complete loss of vitellogenin m R N A activity (Fig. 2C). Moreover, presence of 60 mM diethylpyrocarbonate in the phenol extraction of the post-chromatin fraction inactivated over 90% of vitellogenin m R N A activity (Fig. 2D).

Effect o f diethylpyrocarbonate on the overall purification, size and in vitro synthetic activity o f vitellogenin m R N A To determine more precisely the effect of diethylpyrocarbonate on the isolation of fully active intact m R N A we have purified vitellogenin m R N A from

m

C

o

A

o

D

~2

1

2

4

5

8 /ug R N A / 1 0 0 FJI

12

Fig. 2. T h e R N A c o n c e n t r a t i o n d e p e n d e n c e o f t h e w h e a t g e r m cell-free s y s t e m . Specific a c t i v i t y o f vitel]og e n i n m R N A w a s m e a s u r e d f o r several c o n c e n t r a t i o n s o f t o t a l m R N A p r e p a r e d f~om A, h o m o g e n a t e t r e a t e d w i t h y e a s t R N A (6 m g / m l ) a n d h e p a r i n (4 m g / m l ) ; B, h o m o g e n a t e t r e a t e d w i t h 6 0 m M d i e t h y l p y r o c a r b o n a t e , y e a s t R N A (6 m g / m l ) a n d h e p a r i n (4 m g / m l ) ; C, p u r i f i e d v i t e l l o g e n i n m R N A t r e a t e d f o r 1 0 mill w i t h 3 m M d i e t h y l p y r o c a r b o n a t e a t r o o m t e m p e r a t u r e ; D, p r e s e n c e o f 6 0 m M d i e t h y l p y r o c a r b o n a t e in p o s t - c h ~ o m a t i n f r a c t i o n d u r i n g t h e e x t r a c t i o n o f R N A w i t h c h l o r o f o r m - p h e n o l a t r o o m t e m p e r a t u r e . T h e t r a n s l a t i o n p r o d u c t w a s a n a l y z e d b y r a d i o i m m u n o p r e c i p i t a t i o n as o u t l i n e d in M e t h o d s . Each point represents the average of t w o measurements.

345

crude extracts which had been treated with a combination of diethylpyrocarbonate yeast RNA and heparin. By means of affinity chromatography using oligo(dT)-cellulose and sucrose density gradient centrifugation, it was possible to purify vitellogenin mRNA approximately 700-fold with a recovery of 30%

A E

3

28 S

18 S

30 20 15

i

~o~ o 7 2

4 6 8 cm Fig. 3. E l e c t r o p h o r e s i s o n 2.3% p o l y a c r y l a m i d e - s o d i u m d o d e c y l s u l f a t e gels o f R N A f r a c t i o n s c o l l e c t e d f r o m sucrose gradient. Total m R N A of post oligo(dT)-cellulose was separated on sucrose density gradient as d e s c r i b e d in M e t h o d s . F r a c t i o n s 1, 2, 3 f r o m s u c r o s e g r a d i e n t s (inset) w e r e p r e c i p i t a t e d w i t h e t h a n o l a n d u p o n c e n t r i f u g a t i n n R N A w a s d e r m t t w e d w i t h 90% f o r m a m i d e a n d a n a l y z e d o n gels as d e s c r i b e d in M e t h o d s . T h e l o w e r p a n e l r e p r e s e n t s R N A f r o m f r a e t i o n 1 w h i c h h a d b e e n r e e h r o m a t o g r a p h e d o n oligo( d T ) - c e l i n l o s e c o l u m n . F r a c t i o n s 1, 2, 3 w e r e t e s t e d in a w h e a t g e r m cell-free s y s t e m a n d h a d a specific a c t i v i t y o f 3 8 0 0 0 , 3 0 0 0 0 a n d 2 4 0 0 0 d p m / ~ g R N A , r e s p e c t i v e l y . As m a r k e r s , c h i c k e n r i b o s o m a l R N A w e r e u s e d : 18 S r R N A (M r = 0 . 7 • 1 0 5 ) a n d 2 8 S r R N A M r = 1 . 5 8 • 106 [ 3 1 ] .

346 T A B L E II PURIFICATION OF VITELLOGENIN mRNA. P u r i f i c a t i o n of v i t e l l o g e n i n m R N A f r o m c h i c k e n w h i c h h a d r e c e i v e d a single i n j e c t i o n of e s t r a d i o l is s h o w n . T h e i n d i v i d u a l s t e p s o f p u r i f i c a t i o n are d e s c r i b e d in M e t h o d s . Specific a c t i v i t y of v i t e l l o g e n i n m R N A was m e a s u r e d as p r e v i o u s l y d e s c r i b e d [ 4 ] . E a c h v a l u e r e p r e s e n t s t h e a v e r a g e of t w o m e a s u r e m e n t s minus the radioactivity of the i m m u n o p r e c i p i t a t e of the control incubated without exogenous m R N A . T o t a l a n d large p o l y s o m e s w e r e o b t a i n e d a f t e r 1 8 0 m i n a n d 70 rain c e n t r i f u g a t i o n of liver supern a t a n t f r a c t i o n o v e r 40% s u c r o s e , r e s p e c t i v e l y . P u r i f i c a t i o n step

RNA ( # g / g liver)

Vitellogenin m R N A (cpm/pg RNA)

Total activity ( c p m / g liver)

Total polysomes Large polysomes Oligo(dT)-cellulose Sucrose gradient Oligo(dT)-cellulose

6500 1500 40 6 3

110 421 8 530 37 4 8 4 76 2 2 5

715 631 345 224 228

000 500 465 904 675

20 1000

15

10 80O

E ~

8 6

600

B

C 0

4

U

31

~ ,~o

I

o

v-

2

r,

2:200

M

O0000eaoooOo OOeoPaO Oo o oOOog a Ooooag QOOaoao oooaeO° •

i'"" 1

O0

•

"T " ' " ' " 2

•

t

ea

3

""

000

oaOOOoeOOoo

J

I

4

5

°°°°

oo aao

F 6

• °°qP aooo

cm Fig. 4. P o l y a c r y l a m i d e gel e l e c t r o p h o r e s l s o f t h e in v i t r o t r a n s l a t i o n p r o d u c t o f v i t e l l o g e n i n m R N A in r a b b i t r e t i c u l o c y t e l y s a t e s y s t e m . T h e l a b e l e d p o l y p e p t i d e s s y n t h e s i z e d in v i t r o w e r e c o - i m r n u n o p r e c i p i t a t e d w i t h c a r r i e r v i t e l l o g e n i n as d e s c r i b e d in M e t h o d s a n d t h e r e s u l t i n g p r e c i p i t a t e w a s a n a l y z e d o n 5% p o l y a c r y l a m i d e s o d i u m d o d e c y l s u l f a t e gels. A, r e p r e s e n t s t h e t o t a l 3 H - l a b e l l e d p r o t e i n s m a d e in t h e p r e s e n c e o f v i t e l l o g e n i n m R N A a n d B, t h e i m m u n o p r e c i p i t a b l e 3 H - l a b e l l e d p r o t e i n s w i t h anti-lipovitellin. Gels w e r e c a l i b r a t e d w i t h p a n c r e a t i c d e o x y r i b o n u c l e a s e (M r = 31 0 0 0 ) h e a v y c h a i n o f lipovitellin [ 3 2 ] (M r = 1 3 5 0 0 0 ) .

347 (Table II). Electrophoresis on polyacrylamide gels showed that the vitellogenin m R N A had a molecular weight of 2.4 • l 0 s (Fig. 3, b o t t o m panel}, in agreement with previous findings [4]. Upon separation of total m R N A on sucrose density gradient most of the vitellogenin m R N A was located at the leading edge of the 28 S rRNA peak (Fig. 3), thus permitting the use of a sucrose density gradient as a preparative step in the purification of the mRNA. As shown above, viteUogenin m R N A was fully active upon treatment of the crude homogenate with diethylpyrocarbonate in the presence of yeast R N A and heparin. The purified vitellogenin m R N A can be translated in vitro into a major peptide of molecular weight 220 000 and this peptide can be precipitated with specific anti-lipovitellin (Fig. 4). Discussion In the majority of cases where diethylpyrocarbonate was used as an inactivator of ribonucleases intact RNA was obtained, however very often it lacked of its biological activity [5,14,15]. A few exceptions have been reported; for example, tRNA with amino acid-accepting activity was isolated from human placenta in the presence of diethylpyrocarbonate [25] and Poly(A)containing m R N A from mouse tissue was isolated from polysomes in the presence of diethylpyrocarbonate and the m R N A was subsequently used for in vitro synthesis of complementary DNA [26]. Similarly, Wahli et al. [27] reported the isolation of Xenopus vitellogenin m R N A in the presence of diethylpyrocarbonate and the m R N A was used for the synthesis of cDNA. However, in none of these reports was it shown that the m R N A isolated in the presence of diethylpyrocarbonate had retained its capacity to direct the translation of a full size polypeptide in vitro. Since the rate constant of acylation of histidine residues from proteins is several orders of magnitude higher than the reaction with nucleic acids was it possible to select conditions where nucleic acids in the presence of a vast excess of proteins would react slowly, if at all, with diethylpyrocarbonate. Using diethylpyrocarbonate, at a concentration of 60 mM pH 6.5 and 30°C we found maximum inhibition of liver nucleases. Increasing the concentration of diethylpyrocarbonate (data not shown) did n o t result in higher inactivation of ribonucleases. These results are in agreement with observations of Melchior and Fahrney [28] who found that 5--10% of pancreatic ribonuclease activity resisted inactivation by diethylpyrocarbonate. When diethylpyrocarbonate was used as the sole inhibitor of ribonuclease we routinely had a much lower recovery in polysomes than in other experiments where heparin and/or yeast RNA were used (Table I). The apparent failure to get polysomes is consistent with the observations that diethylpyrocarbonate in a concentration-dependent manner effects the dissociation of ribosomal particles into subunits [15,29,30]. This unwanted side effect of diethylpyrocarbonate could be fully prevented by adding yeast R N A to the liver homogenate. The highest specific activity in vitellogenin m R N A was always obtained when diethylpyrocarbonate-yeast R N A were combined with heparin. Since at 0°C the reactivity of diethylpyrocarbonate with proteins is about 9 times slower than at 30°C [14], it is conceivable that the effect of heparin is to inhibit nucleases while they are being slowly inactivated by diethylpyrocarbonate. Our results

348 also suggest that the sequence of addition of diethylpyrocarbonate during the isolation of vitellogenin m R N A is very important. For example, presence of 60 mM diethylpyrocarbonate in extraction of post-chromatin fraction with sodium dodecyl sulfate-phenol chloroform at room temperature resulted in an almost complete loss of vitellogenin m R N A activity (Fig. 2D). Moreover, purified vitellogenin m R N A treated at room temperature for 10 min with 3 mM diethylpyrocarbonate was completely inactive in a cell-free system (Fig. 2C). Vitellogenin m R N A purified from crude homogenate in the presence of 60 mM diethylpyrocarbonate has a molecular weight (Fig. 3) comparable to that obtained with polysomes isolated by specific indirect immunoprecipitation with anti-lipovitellin [4]. In addition, when vitellogenin m R N A was tested in a cell-free system the translation product had a molecular weight of about 220 000, thus suggesting that diethylpyrocarbonate under our experimental conditions did n o t modify the ability of the m R N A to direct the synthesis of full size vitellogenin. References 1 G e r h n g e r , P., Le Meur, M.A., Clavert, J. a n d Ebel, J.P. ( 1 9 7 3 ) Bioehimie 55, 2 9 7 - - 3 0 7 2 M o r t o n , B., Nwizu, C., H e n s h a w , E.C., Hixsch, C.A. a n d H i a t t , H.H. ( 1 9 7 5 ) Biochim. Biophys. A e t a 395, 28--40 3 A b , G., R o s k a m , W.G., Dijkstra, J., Mulder, J., Willems, M., van der Ende, A. a n d G r u b e r , M. ( 1 9 7 6 ) Biochim. Biophys. A c t a 4 5 4 , 6 7 - - 7 8 4 J o s t , J.P. a n d Pehling, G. ( 1 9 7 6 ) Eur. J. B i o c h e m . 66, 3 3 9 - - 3 4 6 5 R h o a d s , R.E., M c K n i g h t , G.S. a n d S c h i m k e , R.T. ( 1 9 7 3 ) J. Biol. Chem. 2 4 8 , 2 0 3 1 - - 2 0 3 9 6 Haines, M.E., C a r e y , N.H. a n d Palmiter, R.D. ( 1 9 7 4 ) Eur. J. B i o c h e m . 43, 5 4 9 - - 5 6 0 7 Steele, W.J., O k a m u r a , H. a n d Busch, H. ( 1 9 6 5 ) J. Biol. C h e m . 2 4 0 , 1 7 4 2 - - 1 7 4 9 8 B o e d t k e r , H. ( 1 9 6 8 ) M e t h o d s in E n z y m o l o g y 12B, 4 3 2 - - 4 3 3 9 S t e r n , H. ( 1 9 6 8 ) M e t h o d s in E n z y m o l o g y , 12B, 1 0 0 - - 1 1 2 10 L i e h n h a r d , G.E., Secemski, I.I., K o e h l e r , K.A. a n d L i n d q u i s t , R.N. ( 1 9 7 1 ) Cold Spring H a r b o r Syrup. Q u a n t . Biol. 36, 4 5 - - 5 1 11 G r a y , J.C. ( 1 9 7 4 ) Arch. B i o c h e m . Biophys. 163, 3 4 3 - - 3 4 8 12 S h o r t m a n , K. ( 1 9 6 1 ) Biochim. Biophys. A c t a 51, 3 7 - - 4 9 13 Blobel, G. a n d P o t t e r , V.R. ( 1 9 6 6 ) Proc. Natl. A c a d . Sci. U.S. 55, 1 2 8 3 - - 1 2 8 8 14 E h r e n b e r g , L., F e d o r c s a k , I. a n d S o l y m o s y , F. ( 1 9 7 6 ) Progress in Nucleic A c i d R e s e a r c h a n d Moleculax Biology, 16, 1 8 9 - - 2 6 2 15 A n d e r s o n , J.M. a n d K e y , J.L. ( 1 9 7 1 ) Plant Physiol. 4 8 , 8 0 1 - - 8 0 5 16 Bantle, J . A . , Maxwell, I.H. a n d H a h n , W.E. ( 1 9 7 6 ) Anal. B i o c h e m . 72, 4 1 3 - - 4 2 7 17 L o e n i n g , U.E. ( 1 9 6 9 ) B i o c h e m . J. 1 1 3 , 1 3 1 - - 1 3 8 18 Palmiter, R.D. ( 1 9 7 3 ) J. Biol. C h e m . 2 4 8 , 2 0 9 5 - - 2 1 0 6 19 J o s t , J.P. a n d Pehling, G. ( 1 9 7 6 ) Eur. J. B i o c h e m . 62, 2 9 9 - - 3 0 6 20 Palmiter, R.D., O k a , T. a n d S c h i m k e , R.T. ( 1 9 7 1 ) J. Biol. C h e m . 2 4 6 , 7 2 4 - - 7 3 7 21 Bergink, E.W. a n d Wallace, R.A. ( 1 9 7 4 ) J. Biol. C h e m . 2 4 9 , 2 8 9 7 - - 2 9 0 3 22 R o b e r t s , B.E. a n d P a t e r s o n , B.M. ( 1 9 7 3 ) Proc. Natl. A c a d . Sci. U.S. 70, 2 3 3 0 - 2 3 3 4 23 M / / e n p ~ , P.H. a n d Bernfield, M.R. ( 1 9 6 9 ) B i o c h e m i s t r y 8, 4 9 2 6 - - 4 9 3 5 24 L e p p l a , S.H.0 B j o r a k e r , B. a n d B o c k , R.M. ( 1 9 6 8 ) M e t h o d s E n z y m o l . 12B, 2 3 6 - - 2 4 0 25 A b a d o m , P.N. a n d Elson, D. ( 1 9 7 0 ) Biochim. B i o p h y s . A c t a 199, 5 2 8 - - 5 3 1 26 Y o u n g , B.D., Birnie, G.D. a n d Paul, J. ( 1 9 7 6 ) B i o c h e m i s t r y 15, 2 8 2 3 - - 2 8 2 9 27 Wahli, W., Wyler, T., Weber, R. a n d R y f f e l , G.U. ( 1 9 7 6 ) Eur. J. Biochem. 66, 4 5 7 - - 4 6 5 28 Melchior, W.B. and Fahrney, D. ( 1 9 7 0 ) Biochemistry 9, 2 5 1 - - 2 5 8 29 WeUer, D.L., H e a n e y , A., F r a n c e s c h i , R.T., B o u d r e a u , R.E. a n d S h a w , D.E. ( 1 9 7 3 ) A n n . N.Y. A c a d . Sci. 2 0 9 , 2 5 8 - - 2 8 0 3 0 L o n s d a l e , D.M. a n d B o u l t e r , D. ( 1 9 7 3 ) P h y t o c h e m i s t r y 12, 3 9 - - 4 1 31 A t t a r d i , G. a n d A m a l d i , F. ( 1 9 7 0 ) A n n u . Rev. Bioehem. 39, 1 8 3 - - 2 2 6 3 2 J o s t , J.P., Pchling, G. a n d Baca, O.G. ( 1 9 7 5 ) B i o c h e m . B i o p h y s . Res. C o m m u n . 6 2 , 9 5 7 -965