Cell l)ifferenliation, ,1 (1975) 113--122 © North-Holland Publishing Company, Amsterdam - Printed in The Netherlands
M U R I N E M A M M A R Y GLAND RNA D I R E C T E D S Y N T H E S I S O F C A S E I N IN A H E T E R O L O G O U S C E L L - F R E E P R O T E I N S Y N T H E S I S S Y S T E M
P.M. TERRY, R. GANGULY, E.M. BALL anct M.R. BANER,JEE 7"umor Biology Laboratory, School of Life Sciences, University o f Nebraska-Lincoln, Lincoln, Nebraska 68503, U S.A.
Accepted 20 February 1975
A cell-free protein synthesis system derived from Ehrlich ascites tumor cell ribosomes ($30) plus rabbit reticulocyte t R N A was developed and the activity of the system was dependent on rabbit reticulocyte ribosomal salt (0.5 M KCI) wash factors. The exogenous mRNAs from BALB/c mouse liver and the mammary gland were translated with a high efficiency in this heterologous cell-free system. Furthermore, the RNA from the lactating mammary gland faithfully directed the synthesis of casein. The presence of mouse casein in the reaction product was identified by radioimmunopreeipitation with mouse casein antiserum, co-electrophoresis of the reaction product and mouse casein in urea-polyacrylamide gel anti by electrophoresis in sodium dodecyl sulfate (SDS) polyacrylamide gel. The major portion of the lactating mammary gland RNA directed synthesis of the milk protein in the cell-free system appearecl to he analogous to a s casein.
D e v e l o p m e n t of t h e i m m a t u r e m a m m a r y gland involves initial m o r p h o g e n e t i c c h a n g e s o f t h e p a r e n c h y m a p r o d u c i n g t h e a l v e o l a r s t r u c t u r e s followed by functional differentiation of these secretory units into lactation. T h e r e s u l t s o f p r e v i o u s s t u d i e s in this a n d o t h e r l a b o r a t o r i e s i n d i c a t e t h a t f u n c t i o n a l d i f f e r e n t i a t i o n o f t h e m o u s e m a m m a r y p a r e n c h y m a is a s s o c i a t e d w i t h i n c r e a s e d s y n t h e s i s o f r a p i d l y l a b e l e d R N A , o c c u r r e n c e of r o u g h e n d o p l a s m i c r e t i c u l u m , i n d u c t i o n o f s p e c i f i c m i l k p r o t e i n s (Mills et al., 1 9 7 0 ; T u r k i n g t o n , 1 9 7 0 ; B a n e r j e e et al., 1 9 7 1 a , b , 1 9 7 3 ; T o p p e r , 1 9 7 0 ; Rivera, 1 9 7 4 ) . In this r e p o r t we p r e s e n t t h e r e s u l t s o f a s t u d y on m e s s e n g e r R N A ( m R N A ) a c t i v i t y o f t h e l a c t a t i n g m o u s e m a m m a r y gland p r o d u c i n g a s p e c i f i c m i l k p r o t e i n in a h e t e r o l o g o u s cell-free p r o t e i n s y n t h e s i z i n g s y s t e m . MATERIALS AND METHODS Animals
N u l l i p a r o u s , 6 - - 8 w e e k o l d f e m a l e B A L B / c m i c e w e r e b r e d in o u r l a b o r a t o r y a n d l a c t a t i n g m a m m a r y g l a n d s a n d liver o f 6 - 1 0 d a y p o s t - p a r t u m m o t h e r s were used f o r R N A p r e p a r a t i o n .
11t
Isolation of RN..t RNA was e x t r a c t e d at r o o m t e m p e r a t u r e according to a slightly modified p r o c e d u r e previously described by Parish et al. ( 1 9 6 6 ) and Banerjee el al. (1973). The minced tissue was h o m o g e n i z e d in 10 vol of a m i x t u r e of 65; t~-aminosalisylate, 1 C; s o d i u m t r i i s o l ) r o p y l n a p t h a h m e s u l p h o n a t e , 6% 2-butanol and 151 macaloid. An equal v o l u m e o f p h e n o l : m-cresol : 8 h y d r o x y q u i n o l i n e : water (500 : 7 0 : 0 . 5 : 55) m i x t u r e was added to the h o m o g e n a t e , the m i x t u r e was shaken at r o o m t e m p e r a t u r e for 2 0 - - 3 0 rain and the a q u e o u s phase was collected by c e n t r i f u g a t i o n ( 1 2 , 0 0 0 g, 10 rain). The interphase was r e e x t r a c t e d at r o o m t e m p e r a t u r e with c h l o r o f o r m and isoamyl-alcohol (25 : 1, v/v). The p o o l e d a q u e o u s t)hase was made 3% with respect to NaCl and RNA was precipitated with 2 vol 95C~ ethanol for 1 0 - - 1 2 hr at - - 2 0 ° C . RNA c o l l e c t e d by c e n t r i f u g a t i o n was dissolved in a small v o l u m e o f 0.1 M s o d i u m acetate and then adjusted to ; / M with respect to NaCl and stored at .I°C for 15 hr. The p r e c i p i t a t e d RNA, collected hy centrifugati()n (27,000/4, 10 min) was washed in "l M s o d i u m a c e t a t e (pH 6) to r e m o v e NaC1, residual DNA and glycogen. The precipitate was rinsed with 75~)~ ethanol c o n t a i n i n g 25; NaAc, centrifuged, resuspended in 95% ethanol (--20:'C), c e n t r i f u g e d , vacuunl desi('('ate(I, dissolved in water and stored at -20¢:C for s u b s e q u e n t analysis.
[h'eporation of ribosomes, factors and tRNA The cell-free protein synthesis system was derived from ascites t u m o r cell ribosomes, rabbit r e t i c u l o c y t e factors and t R N A . Ehrlich ascites t u m o r cells {line E182), m a i n t a i n e d in our l a b o r a t o r y by serial intraperitoneal passages in Swiss mice were used as a source o f ribosomes. The ascites fluid containing the t u m o r was collected m p r e c o o l e d tubes asceptically by peritoneal perfusion using a Pasteur pipet. The s u b s e q u e n t p r o c e d u r e s for p r e p a r a t i o n o f the r i b o s o m e s were essentially similar to those descrihed by Mathews et al. ( 1 9 7 0 ) and McI)owell et al. (1972). The ascites fluid was filtered t h r o u g h cheese cloth, mixed with 5 vol o f cold wash b u f f e r [35 mM Tris--HCl (pH 7.5), 146 mM NaCI, 2 mg/ml glucose] and the t u m o r cells were s e d i m e n t e d t)y c e n t r i f u g a t i o n ( 8 0 g for 5 min). The pellet was washed sew~,ral times in the same b u f f e r to r e m o v e red blood {:ells. After final wash the s u p e r n a t a n t was discarded and the t u m o r cells were pelleted for 10 rain at 2 0 0 0 g . The t u m o r cell pellet was then resuspended (3 ml/ml packed cells) in the h o m o genizing b u f f e r [10 mM H E P E S (pH 7.5), 15 mM KCI, 1 mM MgAc~, 6 mM ~5-mereaptoethanol], allowed to swell for 10 min and gently h o m o g e n i z e d I)y 4 0 - - 5 0 strokes in a hand o p e r a t e d D o u n c e h o m o g e n i z e r . Isotonicity of the h o m o g e n a t e was restored by i m m e d i a t e l y adding 0.1 vol o f 10 X H E P E S b u f f e r [ 2 0 0 mM HEPES (pH 7.5), 1 . 2 M KC1, 50 mM MgAc~, 60 mM ~m e r c a p t o e t h a n o l ] . The h o m o g e n a t e was centrifuged for 20 min at 3 0 , 0 0 0 g and the s u p e r n a t a n t (S~,,) was collected. To the S ~ , 1 mM ATP, 0.2 mM
115
GTP, 4 mM 2-phosphoenol pyruvic acid (PEP), 7.5 IU/ml PEP kinase and a mixture of unlabeled amino acids (70 #M each) were added. The mixture was incubated at 37°C for 40 min and centrifuged ( 2 0 , 0 0 0 g , 10 min). The supernatant was then passed through G-25 Sephadex column (2.6 × 40 cml that had been equilibrated with 1 X ttEPES buffer. The contents of the fraction tubes having greater than 15 absorbance (260 nm) units/ml were pooled and stored (--196 ° C) for subsequent use as ribosomes ( $ 3 . ) . Factors were prepared by extracting rabbit reticulocyte ribosomes I S ) o ) with 0.5 M KC1 (Woodley et al., 1974) and t RN A was obtained also from rabbit reticulocytes as described by Bose et al. (1974).
Cell-free protein synthesis assay mixture The standard assay mixture contained in a total volume of 0.075 ml was c o mp o s ed of 18 mM Tris--HC1 (pll 7.5), 22 mM NaCl, 97 mM KC1. 3 mM Mg ÷*, 0.2 mM dithiothreitol (DTT), 1 mM ATP, 0.2 mM GTP, 4 mM PEP, 4 IU/ml pyruvate kinase, 67 pM each of 19 unlabeled amino acids, 6.66 pCi/ml 4,5-~H-leucine (67.6 Ci/mmole), 57 /ag/ml unfractionated tRNA. To this mixture 0.54 absorbance (260 nm) units $30 ribosomes, 0.23 absorbance (280 nm) units of rabbit reticulocyte ribosomal 0.5 M KC1 wash (factors) and various concentrations of m a m m a r y gland or liw~r RNA were added. Incubation was done at 30°C for 40 min.
Casein assay The p r o d u c t of the cell-free system was assayed for mouse casein by an appropriately modified immunoprecipitation procedure similar to that used for d etectio n of ovalbumin by Means et al. (1972). Mouse casein was prepared by Ca**-rennin precipitation of BALB/c mouse milk (Jeurgens et al., 1965) and rabbit antiserum to casein was induced by injecting mouse milk casein into rabbits according to the procedure described by Feldman et al. (1970) and Feldman (1974). The casein antiserum (adsorbed 1 : 1 with mouse serum) and tested by immunodiffusion in 0.5% agar buffered with 0.01 M neutral phosphate. Zones of precipitation were observed with mouse milk, Ca**-rennin precipitate (casein) of mouse milk extract of lactating m a m m a r y tissue. No reaction was observed with virgin m a m m a r y gland, liver e x t ract or mouse serum. The a n t i b o d y was also tested by an indirect radioi m m u n o p r e c i p i t a t i o n procedure. The casein antiserum was first incubated at 4°C with various antigens overnight, the ' : 51 labeled mouse casein was then added to the tubes and 2 hr later samples were assayed. Mouse casein was labeled with ~: sI according to the methods of Bale et al. (1966). Binding of 251 casein to the ant i body was negligible in tubes preincubated with milk and Ca*'-rennin precipitate but ~251 casein binding to the antiserum was very high in tubes with virgin m am m ar y gland and liver extracts, mouse serum or rennin as antigens, indicating a specific affinity of the antisera for
116
mouse casein. A cletailed a c c ount of this indirect immunoprecipitation procedure for mouse casein will be reported elsewhere. For detcction of casein in the p r o d u c t o f the cell-free system, 20 pl of anticasein serum was added in a small tube, followed by 20/al 5,% sodium d e o x y c h o l a t e (w/v), 12 ~1 .1% Triton X-100 (v/v), 30 pl reaction mixture, 1 pg mouse milk (skimmed) carrier plus buffer containing 1 mM NaPO4 (pH 7.5) and 15 mM NaCI to a final volume of 96 ~1. The mixture was allowed to stand for 30 min at r oom temperature, 1 ml of 'wash' buffer [10 mM NaPOa (pH 7.5), 150 mM NaCI, 35~ Triton X-100, and 10 mM unlabeled leucine] was then added and contents of the tubes were thoroughly mixed. The precipitate was collected on an 0.45 p Millipore filter, rinsed with 20 ml of the 'wash' buffer, dried and counted for radioactivity.
Urea polyacrylamide gel ele(.trophoresis Mouse milk carrier prepared by pll 4.6 precipitation of the milk was dissolved in imidazole (KC1, 0.15 M; NaPO4, 0.004; CaC12, 0.01 M; imidazole, 0.01 M; pH 6.8) bufh,r. 2 mg of mouse milk carrier was added to 370 pl of cell-free reaction mixture and the solution was made 0.01 M with respect to CaC12 and rennin was added to 0.85 mg/ml. The solution was then incubated at 37°C for 30 min; tile precipitate, collected by centrifugation, was washed in imidazole buffer, 5~ trichloroacetic acid (TCA), and dissolved m 7 M urea. 1 0 0 / l g samples per gel were then subjected to electrophoresis in 7V~ polyacrylamide gel in presence of 7 M urea (pH 8.9} as previously descrit)cd by Feldman et al. (1971).
SDS polyaco, lamide gel electrophoresis The mouse casein antibody and the antigen complex obtained by the m e t h o d described above (see casein assay) was dissolved in 50 gl of 1% sodium dodecyl sulfate (SDS) and 50 pl of 2 mg/ml skimmed mouse milk in 1% SDS was added to the solution. The mixture was then solubilized by heating at 70°C for 2 min. After addition of /3-mercaptoethanol (1% final c o n c e n t r a t i o n in 1% SDS) the solution was made 7 ~ with respect to sucrose and 0.02 mg/ml b r o m o p h e n o l blue and the samples were subjected to electrophoresis as described by Weber et al. (1969). Protein determinations were done by the methods of Lowry et al. (1951) using bovine casein as standard. All operations were carried out at 0--4°C e x c e p t where mentioned otherwise and radioactivity of the samples was determined by liquid scintillation s p e c t r o m e t r y in a Beckman LS 350 counter. RESULTS Data in Table I show the responsiveness of the cell-free protein synthesizing system derived f r om Ehrlich ascites t u m o r ribosomes and rabbit reticulo-
117 TABLE I Requirements for translation of exogenous mRNA in a cell-free protein synthesis system, prepared from Ehrlich's ascites tumor cells.
Exogenous RNA
Assay condition
cpm/0.1 ml reaction mixture
None None Liver Mammary gland None Liver Mammary gland
Reaction mixture + tRNA + S.~(~ + RNA + S~(j + RNA + S~o + F + S~) + RNA + F + S~o + RNA + F + S.~(,
390 5,600 15,200 18,000 44,200 101,600 119,440
Standard reaction mixture in 0.075 ml volume as described in Materials and Methods were used. RNAs were prepared from 6 l ( ) d a y lactating mice. The optimum concentrations of 6 u g liver RNA or 1 3 u g mammary gland RNA per 0.1 ml were used. Higher concentrations of RNA were inhihitory in this system. Aliquots of the reaction mixture were assayed for trichloroacetic acid insoluble :~tt-leucine radioactivity by the filter disc method of Woodley et al. (1974). Data represent typical values of 5 10determinations. F, rahhit reticulocyte ribosome salt (0.5 M KCI) wash factors, purified hy DEAE cellulose column chromatography according to Woodley et al. (197,1).
c y t e f a c t o r s . As m e a s u r e d b y "~H - l e u c i n e i n c o r p o r a t i o n i n t o an a c i d i n s o l u b l e m a t e r i a l , l i v e r o r m a m m a r y g l a n d m R N A d i r e c t e d p r o t e i n s y n t h e s i s in t h e cell-free system remained low when the rabbit reticulocyte factors were not i n c l u d e d in t h e r e a c t i o n m i x t u r e . T h e a c t i v i t y w a s h i g h e r w h e n t h e r e a c t i o n w a s c a r r i e d o u t w i t h a s c i t e s cell r i b o s o m e s a n d t h e r e t i c u l o c y t e f a c t o r b u t n o added mRNA. This increased activity presumably represents translation of the residual endogenous mRNA. Addition of the exogenous RNA from mouse liver or mammary gland produced more than 2-fold stimulation of ~*H - l e u c i n e i n c o r p o r a t i o n i n t o a n a c i d i n s o l u b l e m a t e r i a l . T h i s d e m o n s t r a t e s that the present cell-free protein synthesis system with Ehrlich ascites tumor c e l l r i b o s o m e s is d e p e n d e n t o n r a b b i t r e t i c u l o c y t e f a c t o r s a n d t h e s y s t e m is capable of effective translation of exogenous mRNA. T h e e x o g e n o u s m R N A d i r e c t e d p r o d u c t o f t h e c e l l - f r e e s y s t e m was t h e n a n a l y s e d f o r a s p e c i f i c m i l k - p r o t e i n , c a s e i n a n d t h e r e s u l t s a r e s h o w n in T a b l e I1. E v i d e n c e o f c a s e i n s y n t h e s i s w a s a b s e n t in ' t h e p r o d u c t o f t h e c e l l - f r e e s y s t e m w i t h n o a d d e d e x o g e n o u s m R N A a n d n o c a s e i n s y n t h e s i s was p r e s e n t in t h e r e a c t i o n p r o d u c t d i r e c t e d b y l i v e r m R N A . B u t R N A o f t h e l a c t a t i n g mammary gland directed the synthesis of a product which was precipitable by a specific antiserum to mouse milk casein. This observation indicates that t h e h e t e r o l o g o u s c e l l - f r e e p r o t e i n s y n t h e s i s s y s t e m d e r i v e d f r o m E h r l i c h asc i t e s t u m o r cell r i b o s o m e s a n d r a b b i t r e t i c u l o c y t e f a c t o r s is c a p a b l e o f f a i t h -
llH TABLE 1I Messenger activity of different RNA preparations on synthesis of casein in an Ehrlich ascites tumor cell ribosome protein synthesis system. Source of RNA .
.
.
.
.
.
.
.
.
None Liver Mammary gland
Casein epm/0.1 ml reaction mixture .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
0 0 I 1,750
IAwn" and mammary ~laud RNA were prepared from 6 10 day lactating mice. Casein cpm was determined by specific immunoprecipitation of the 31/.leucine labeled product of cell-free syslem and mouse casein antiserum. The l)recipitate was immobilized on a nilrocellulose fiher and counted for radioa('tivity as described in Materials and Methods. Normal rabbit serum (preimmune) background cpm(320- 1000)/0.1 ml reaction mixture were suhtracted from all casein counts, l)ala represent typical wdues of 3--6 determinations.
fully translating the m u r i n e m a m m a r y gland m R N A for casein, a specific milk p r o t e i n . T h e l a c t a t i n g m a m m a l 5 , gland and liver m R N A d i r e c t e d p r o d u c t m the cell-free s y s t e m was also a n a l y s e d by u r e a - 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 i s a n d t h e results are s h o w n in Fig. 1. C o n s i s t e n t with earlier r e p o r t s ( F e l d m a n et al., 1 9 7 1 ) m o u s e milk carrier casein (Ca++-rennin p r e c i p i t a t e ) a f t e r ureap 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 i s resolved into distinct a b s o r b a n c e b a n d s and the p r e s e n t p e a k s 3, 4, and 5 were c o r r e s p o n d i n g to similar regions o f the gel, p r e v i o u s l y identified as c o m p o n e n t s o f m o u s e milk casein. A high level o f r a d i o a c t i v i t y o f the m a m m a r y gland R N A d i r e c t e d p r o d u c t o f the cell-free s y s t e m was associated with the a b s o r b a n c e p e a k s 3, 4, and 5. Whereas the r a d i o a c t i v i t y of the liver R N A d i r e c t e d p r o d u c t did n o t s h o w a n y c o r r e s p o n d e n c e with these casein c o m p o n e n t s . These results again d e m o n strate the t r a n s l a t i o n o f the l a c t a t i n g m a m m a r y gland R N A into a specific milk p r o t e i n , casein in a h e t e r o l o g o u s cell-free p r o t e i n s y n t h e s i z i n g s y s t e m . As s h o w n in Fig. 2, casein, s y n t h e s i z e d in the cell-free s y s t e m , was t h e n c h a r a c t e r i z e d by e l e c t r o p h o r e s i s in p o l y a c r y l a m i d e gel c o n t a i n i n g s o d i u m d o d e c y l sulfate (SDS). A f t e r dissociating the antigen- a n t i b o d y c o m p l e x by h e a t the s o l u t i o n was s u b j e c t e d to e l e c t r o p h o r e s i s . The r a d i o a c t i v e p r o t e i n m i g r a t e d in the gel as a p r o n o u n c e d p e a k c o r r e s p o n d i n g to the light chain ( L-chain ) o f r a b b i t "),-globulin. F u r t h e r m o r e , similar e l e c t r o p h o r e s i s of m o u s e milk casein in parallel gels also resolved into s h a r p a b s o r b a n c e p e a k corres p o n d i n g to the p e a k o f the light chain. It is r e a s o n a b l e , t h e r e f o r e , to conclude t h a t the high level o f r a d i o a c t i v i t y associated with the light chain represenks a c o m p o n e n t of m o u s e milk casein actively s y n t h e s i z e d in the cell-free s y s t e m . T h e m o l e c u l a r weight o f the light chain of 7-globulin is 2 3 , 5 0 0 (Weber et al., 1969), and the m o l e c u l a r weight o f the b o v i n e ~
119
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Fig. 1. U r e a - p o l y a e r y l a m i d e gel e l e c t r o p h o r e s i s of the p r o d u c t of cell-free p r o t e i n s y n t h e sis s y s t e m . At the end o f the reaction, 2 m~ of m o u s e milk carrier was a d d e d to the r e a c t i o n m i x t u r e , casein was p r e c i p i t a t e d by Ca++-rennin. The p r e c i p i t a t e was then dissolved in 7 M urea, 100/.tg samples were layered on 71~ a e r y l a m i d e gel and duplicate gels were s u b j e c t e d to e l e c t r o p h o r e s i s (Davis, 1964) for 2..10 rain at 3 m A per gvl. One eel was stained with buffalo black, a b s o r b a n e e (400 tam) was measured in a Gitford gel scanner. The s e c o n d gel was s e c t i o n e d (1 ram) with a Miekel gel slicer. The sections were covered with 0.5 ml Nuclear Chicago Soluhilizer (NCS) in liquid scintillation vials. i n c u b a t e d at 50°C overnight and swelled s e c t i o n s were then c o u n t e d for radioactivity. Note t h a t 3 H-leucine radioactivity o f only the m a m m a r y gland RNA p r o d u c t c o r r e s p o n d s to a b s o r b a n c e p e a k s 3, 4, a n d 5 o f m o o s e c a s e i n . • • , M a m m a r y gland; • . . . . . A, Liver. Fig. 2. C h a r a c t e r i z a t i o n o f the p r o t e i n s s y n t h e s i z e d in the cell-free s y s t e m by p o l y a e r y l amide-SDS gel e l e e t r o p h o r e s i s . The anticasein p r e c i p i t a t e o f the cell-free p r o d u c t of m a m m a r y gland or liver RNA was collected and treated as descrihed in the e x p e r i m e n t p r o c e d u r e . 100t~g o f the material ( p r o t e i n s ) was layered on p o l y a c r y l a m i d e - S D S gel and s u b j e c t e d to e l e e t r o p h o r e s i s as described by Weber et al. (1969). Duplicate gels were run for each e x p e r i m e n t , one was stained with coomassie blue and a h s o r b a n e e (550 n m ) w a s m e a s u r e d in a Gilford gel scanner. The s e c o n d gel was s e c t i o n e d ( 1 ram) with a Mickel gel slicer, the s e c t i o n s were m o i s t e n e d in a liquid scintillation vial, 10 ml o f .1% NCS (in t o l u e n e - b a s e d scintillation fluid) was a d d e d and i n c u b a t e d at 37°C overnight. The swelled s e c t i o n s were t h e n c o u n t e d in a liquid scintillation system. Note the p r o n o u n c e d radioactivity o f m a m m a r y R N A p r o d u c t in the region o f the light chain o f the )'-globulin ahsorl)ance peak (arrow). Parallel p o l y a c r y l a m i d e - S D S gel e l e c t r o p h o r e s i s o f m o u s e milk casein also s h o w e d a sharp a b s o r b a n c e peak in the n,gion c o r r e s p o n d i n g to the light chain o f the y-globulin. S o m e radioactivity o f the m a m m a r y gland R N A p r o d u c t was also associated with the m i n o r peak o f milk p r o t e i n handing in the area o1' gel s e c t i o n s 19--22. Radioactivity o f the liw~r RNA p r o d u c t did n o t s h o w any association with the a h s o r h a n c e profih'. . . . . . . . . . . . . , M a m m a r y gland; o- o Liver.
( M e r c e i r e t al., 1 9 7 2 ) . T h u s , i n v i e w o f t h e c l o s e n e s s o f the molecular weight and the electrophoretic mobility pattern of the two proteins we assume that the bulk of the lactating m a m m a r y gland m R N A directed synthesis of casein in the present cell-free system represents a~ cornc a s e i n is 2 2 , 9 7 5
120 p o n e n t o f m o u s e casein, the major fraction of the milk protein. This interp r e t a t i o n is c o n s i s t e n t with the observation t h a t m R N A of lactating mammary gland o f the ewe directs d e t e c t a b l e synthesis o f only a s casein in a rabbit r e t i c u l o c y t e lysate protein synthesis system (Gaye et al., 1973). A m o d e s t a m o u n t of radioactivity in the p r o d u c t o f the lactating m a m m a r y gland RNA was also associated with a m i n o r peak of casein c o m p o n e n t in the S D S - p o l y a c r y l a m i d e gel banding in th(, region of gel sections 19- 22 as s h o w n in Fig. 2. I d e n t i f i c a t i o n o f this casein c o m p o n e n t synthesized in the cell-free s y s t e m is n o t clear at this time. DISCUSSION The p r e s e n t results d e m o n s t r a t e that messenger activity o f RNA isolated f r o m the lactating murine m a m m a r y gland is capable o f directing the synthesis o f a specific milk protein, casein in a cell-free p r o t e i n synthesis system. The results also revealed t h a t translation o f the m R N A for as casein in the ascites t u m o r cell r i b o s o m e s y s t e m does n o t require the presence of species specific factors and these observations are in a g r e e m e n t with similar results previously described in o t h e r e u c a r y o t i c systems ( G u r d o n et al., 1971; Mathews et al., 1971; Rhoads et al., 1972; Rosenfeld et al., 1972; Gaye et al., 1973). However, the present findings c o n s t i t u t e the first d e m o n s t r a t i o n t h a t m R N A for a specific milk p r o t e i n (or its c o m p o n e n t s ) can be faithfully translated in a cell-free p r o t e i n synthesis s y s t e m derived f r o m Ehrlich ascites t u m o r cell r i b o s o m e s and rabbit r e t i c u l o c y t e factors. It is not y e t k n o w n which f r a c t i o n o f the p o l y s o m a l RNA of the lactating m a m m a r y gland o f the mouse c o d e for the milk-protein and these studies are in progress. Preliminary results suggest t h a t p o l y s o m a l aggregates of ,1, 6, and 12 ribosomal units o f the lactating m a m m a r y gland are positive in casein m R N A activity. Multiple h o r m o n a l regulation o f the f u n c t i o n a l d i f f e r e n t i a t i o n o f m u r i n e mammary gland is well delineated (Nandi et al., 1961; T o p p e r , 1970; T u r k i n g t o n et al., 1973; Rivera, 1974; Wood et al., 1975), b u t the h o r m o n e or combination o f h o r m o n e s involved in the transcriptional regulation o f milk-protein m R N A y e t needs to be elucidated. It is n o w possible to simulate the biological cycle o f growth, lactation and involution o f the m a m m a r y p a r e n e h y m a in organ c u l t u r e o f the entire gland, by a p p r o p r i a t e l y regulating the h o r m o n a l s u p p l e m e n t a t i o n o f the serum-free s y n t h e t i c m e d i u m (Banerjee et al., 1974; Wood et al., 1975). E x t r a c t i o n and cell-free translation of fractions of RNA f r o m the gland cultivated in m e d i u m with various h o r m o n e s is c u r r e n t l y u n d e r w a y in o u r l a b o r a t o r y and the results should elucidate the mechanisms of i n d u c t i o n o f h o r m o n e m o d u l a t e d milk p r o t e i n m R N A in m a m m a r y differentiation. ACKNOWLEDGEMENTS Supported by a grant from the National Cancer Institute. We thank Prof~,ssor N.K.
121 Gupta of our Chemistry Department for advice and Dr. C. Woodley for help in developing the cell-free system, Mr. M. Loudon and Ms. Linda Washburn for technical assistance and Ms. Naomi Windhorst for seeretarial services. A preliminary report of these results was presented at the 14th Annual Meetings of the American Society of Cell Biology, San Diego, California, November 21 -23, 197,1.
REFERENCES Bale, W.F., R.W. Helmkamp, T.P. Davis, M.J. Izzo, R.L. Goodland, M.R. Contreras and I.L. Sparr: Proc. Soc. Exptl. Biol. Med. 122, .107--,114 (1966). Banerjee, D.N. and M.R. Banerjee: d. Endocr. 56, 145--152 (1973a). Banerjee, D.N., M.R. Banerjee and R.G. Mehta: J. Natl. Cancer Inst. 51, 8.13--8.19 ( 1973b). Banerjee, M.R. and D.N. Banerjee: Exptl. Cell Res. 64, 307--316 (1971a). Banerjee, M.R., F.M. Rogers and D.N. Banerjee: J. Endocr. 50, 281--291 (1971b). Banerjee, M.R., B.G. Wood and L.L. Washburn: J. Natl. Cancer Inst. 53, 1387--1393 (1974). Bose, K.K., N.K. Chatterjee and N.K. Gupta: In: Methods of Enzymology, eds. K. Mohlave and L. Grossman (Academic Press, New York) p. 522 (197.1). Davis, B.: Ann. N.Y. Acad. Sci. 121, 40,1--427 (196,1). Feld man, M. K. : Comp. Biochem. Physiol. t 9A, 127-- 135 ( 1974 ). Feldman, M.K. and R.L. Ceriani: Comp. Biochem. PhysioL 37, 421--427 (1970). Feldman, M.K. and P. llohmann: Int. J. Biochem. 2, 477--480 (1971). Gaye, P., L. Itoudebine and R. Denamur: Bioehem. Biophys. Res. Commun. 51, 637644 (1973). Gurdon, J.B., C.D. Lane, H.R. Woodland and G. Marbaix: Nature 233. 177 182 (1971). Jeurgens, W., F. Stockdale, Y.J. Topper and J. Elias: Proc. Natl. Acad. Sci. U.S. 54, 6 2 9 634 (1965). Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall: J. Biol. Chem. 193, 265- 275 (1951). Mathews, M.B. and A. Korner: Eur. J. Biochem. 17, 328- 338(1970). Mathews, M.B.,J. Osbornand J.B. Lingrel: Nature New Biol. 233, 206- 209 (1971). McDowell, M.J., W.K. Joklik, L. Villa-Komaroff and It.F. Lodish: Proc. Natl. Aead. Sci. U.S. 69, 2649--2653 (1972). Means, A.R., J.P. Cornstoek, G.C. Rosenfeld and B.W. O'Malley: Proe. Natl. Aead. Sei. U.8. 69, 1146--1150(1972). Mercier, J.C., F. Groselaude and B. Ribadeau-Dumas: In: Atlas of Protein Sequences and Strueture, ed. M.O. Dayhoff (Natl. Bioehem. Res. Foundation, Georgetown University, Medical Center, Washington, D.C.) p. D320 (1972). Mills, E.S. and Y.J. Topper: J. Cell Biol..t4, 310 328 (1970). Nandi, S. and tt.A. Bern: Gen. Comp. Endocr. 1, 195 210(1961). Parish, J.G. and K.S. Kirby: Biochim. Biophys. Aeta 129, 554--562 (1966). Rhoads, R.E., G.S. McKnight and R.T. Schimke: J. Biol. Chem. 246, 7 4 0 7 7410 (1971}. Rivera, E.M.: In: Lactogenic Hormones, Fetal Nutrition and Lactation, ed. J.D. Josimorich (Wiley, New York) p. 279 (1974). Rosenfehl, G.C., J.P. Comstock, A.R. Means and B.W. O'Malley: Bioehem. Biophys. Res. Commun. 46, 1695--1703 (1972). Topper, Y.,I.: Reeent Progr. Hormone Res. 26, 287- 308(1970). Turkington, R.W.: J. Biol. Chem. 245, 669(}---6697 (1970). Turkmgton, R.W., J.C. Majumder, N. Kadohama, J.tt. Maelndoe and W.L. Frantz: Recent Progr. Hormone Res. 29, .I17 -455 (1973).
122 Weber, K. a n d M. O s b o r n : , l . Biol. Chem. 2.14,-1.t06 -t.112(1969). Wood, B.G., L.L. Washl)urn. A.S. M u k h e r j e e and M.II. Banerjee: ,I. l~;ndocr. 61 (in t)l'~,ss) ( 19T 5). Woodley, C.I,., Y.C. Chen and N.K. ( ; u p t a : I n : M e t h o d s of Enzymoh)gy>¢,ds. K. Mc>ldav,, and L. (h'¢~sman ( A c a d e m i c Pla,ss, New York ) p. 177 (19';'4).
M U R I N E M A M M A R Y GLAND RNA D I R E C T E D S Y N T H E S I S O F C A S E I N IN A H E T E R O L O G O U S C E L L - F R E E P R O T E I N S Y N T H E S I S S Y S T E M
P.M. TERRY, R. GANGULY, E.M. BALL anct M.R. BANER,JEE 7"umor Biology Laboratory, School of Life Sciences, University o f Nebraska-Lincoln, Lincoln, Nebraska 68503, U S.A.
Accepted 20 February 1975
A cell-free protein synthesis system derived from Ehrlich ascites tumor cell ribosomes ($30) plus rabbit reticulocyte t R N A was developed and the activity of the system was dependent on rabbit reticulocyte ribosomal salt (0.5 M KCI) wash factors. The exogenous mRNAs from BALB/c mouse liver and the mammary gland were translated with a high efficiency in this heterologous cell-free system. Furthermore, the RNA from the lactating mammary gland faithfully directed the synthesis of casein. The presence of mouse casein in the reaction product was identified by radioimmunopreeipitation with mouse casein antiserum, co-electrophoresis of the reaction product and mouse casein in urea-polyacrylamide gel anti by electrophoresis in sodium dodecyl sulfate (SDS) polyacrylamide gel. The major portion of the lactating mammary gland RNA directed synthesis of the milk protein in the cell-free system appearecl to he analogous to a s casein.
D e v e l o p m e n t of t h e i m m a t u r e m a m m a r y gland involves initial m o r p h o g e n e t i c c h a n g e s o f t h e p a r e n c h y m a p r o d u c i n g t h e a l v e o l a r s t r u c t u r e s followed by functional differentiation of these secretory units into lactation. T h e r e s u l t s o f p r e v i o u s s t u d i e s in this a n d o t h e r l a b o r a t o r i e s i n d i c a t e t h a t f u n c t i o n a l d i f f e r e n t i a t i o n o f t h e m o u s e m a m m a r y p a r e n c h y m a is a s s o c i a t e d w i t h i n c r e a s e d s y n t h e s i s o f r a p i d l y l a b e l e d R N A , o c c u r r e n c e of r o u g h e n d o p l a s m i c r e t i c u l u m , i n d u c t i o n o f s p e c i f i c m i l k p r o t e i n s (Mills et al., 1 9 7 0 ; T u r k i n g t o n , 1 9 7 0 ; B a n e r j e e et al., 1 9 7 1 a , b , 1 9 7 3 ; T o p p e r , 1 9 7 0 ; Rivera, 1 9 7 4 ) . In this r e p o r t we p r e s e n t t h e r e s u l t s o f a s t u d y on m e s s e n g e r R N A ( m R N A ) a c t i v i t y o f t h e l a c t a t i n g m o u s e m a m m a r y gland p r o d u c i n g a s p e c i f i c m i l k p r o t e i n in a h e t e r o l o g o u s cell-free p r o t e i n s y n t h e s i z i n g s y s t e m . MATERIALS AND METHODS Animals
N u l l i p a r o u s , 6 - - 8 w e e k o l d f e m a l e B A L B / c m i c e w e r e b r e d in o u r l a b o r a t o r y a n d l a c t a t i n g m a m m a r y g l a n d s a n d liver o f 6 - 1 0 d a y p o s t - p a r t u m m o t h e r s were used f o r R N A p r e p a r a t i o n .
11t
Isolation of RN..t RNA was e x t r a c t e d at r o o m t e m p e r a t u r e according to a slightly modified p r o c e d u r e previously described by Parish et al. ( 1 9 6 6 ) and Banerjee el al. (1973). The minced tissue was h o m o g e n i z e d in 10 vol of a m i x t u r e of 65; t~-aminosalisylate, 1 C; s o d i u m t r i i s o l ) r o p y l n a p t h a h m e s u l p h o n a t e , 6% 2-butanol and 151 macaloid. An equal v o l u m e o f p h e n o l : m-cresol : 8 h y d r o x y q u i n o l i n e : water (500 : 7 0 : 0 . 5 : 55) m i x t u r e was added to the h o m o g e n a t e , the m i x t u r e was shaken at r o o m t e m p e r a t u r e for 2 0 - - 3 0 rain and the a q u e o u s phase was collected by c e n t r i f u g a t i o n ( 1 2 , 0 0 0 g, 10 rain). The interphase was r e e x t r a c t e d at r o o m t e m p e r a t u r e with c h l o r o f o r m and isoamyl-alcohol (25 : 1, v/v). The p o o l e d a q u e o u s t)hase was made 3% with respect to NaCl and RNA was precipitated with 2 vol 95C~ ethanol for 1 0 - - 1 2 hr at - - 2 0 ° C . RNA c o l l e c t e d by c e n t r i f u g a t i o n was dissolved in a small v o l u m e o f 0.1 M s o d i u m acetate and then adjusted to ; / M with respect to NaCl and stored at .I°C for 15 hr. The p r e c i p i t a t e d RNA, collected hy centrifugati()n (27,000/4, 10 min) was washed in "l M s o d i u m a c e t a t e (pH 6) to r e m o v e NaC1, residual DNA and glycogen. The precipitate was rinsed with 75~)~ ethanol c o n t a i n i n g 25; NaAc, centrifuged, resuspended in 95% ethanol (--20:'C), c e n t r i f u g e d , vacuunl desi('('ate(I, dissolved in water and stored at -20¢:C for s u b s e q u e n t analysis.
[h'eporation of ribosomes, factors and tRNA The cell-free protein synthesis system was derived from ascites t u m o r cell ribosomes, rabbit r e t i c u l o c y t e factors and t R N A . Ehrlich ascites t u m o r cells {line E182), m a i n t a i n e d in our l a b o r a t o r y by serial intraperitoneal passages in Swiss mice were used as a source o f ribosomes. The ascites fluid containing the t u m o r was collected m p r e c o o l e d tubes asceptically by peritoneal perfusion using a Pasteur pipet. The s u b s e q u e n t p r o c e d u r e s for p r e p a r a t i o n o f the r i b o s o m e s were essentially similar to those descrihed by Mathews et al. ( 1 9 7 0 ) and McI)owell et al. (1972). The ascites fluid was filtered t h r o u g h cheese cloth, mixed with 5 vol o f cold wash b u f f e r [35 mM Tris--HCl (pH 7.5), 146 mM NaCI, 2 mg/ml glucose] and the t u m o r cells were s e d i m e n t e d t)y c e n t r i f u g a t i o n ( 8 0 g for 5 min). The pellet was washed sew~,ral times in the same b u f f e r to r e m o v e red blood {:ells. After final wash the s u p e r n a t a n t was discarded and the t u m o r cells were pelleted for 10 rain at 2 0 0 0 g . The t u m o r cell pellet was then resuspended (3 ml/ml packed cells) in the h o m o genizing b u f f e r [10 mM H E P E S (pH 7.5), 15 mM KCI, 1 mM MgAc~, 6 mM ~5-mereaptoethanol], allowed to swell for 10 min and gently h o m o g e n i z e d I)y 4 0 - - 5 0 strokes in a hand o p e r a t e d D o u n c e h o m o g e n i z e r . Isotonicity of the h o m o g e n a t e was restored by i m m e d i a t e l y adding 0.1 vol o f 10 X H E P E S b u f f e r [ 2 0 0 mM HEPES (pH 7.5), 1 . 2 M KC1, 50 mM MgAc~, 60 mM ~m e r c a p t o e t h a n o l ] . The h o m o g e n a t e was centrifuged for 20 min at 3 0 , 0 0 0 g and the s u p e r n a t a n t (S~,,) was collected. To the S ~ , 1 mM ATP, 0.2 mM
115
GTP, 4 mM 2-phosphoenol pyruvic acid (PEP), 7.5 IU/ml PEP kinase and a mixture of unlabeled amino acids (70 #M each) were added. The mixture was incubated at 37°C for 40 min and centrifuged ( 2 0 , 0 0 0 g , 10 min). The supernatant was then passed through G-25 Sephadex column (2.6 × 40 cml that had been equilibrated with 1 X ttEPES buffer. The contents of the fraction tubes having greater than 15 absorbance (260 nm) units/ml were pooled and stored (--196 ° C) for subsequent use as ribosomes ( $ 3 . ) . Factors were prepared by extracting rabbit reticulocyte ribosomes I S ) o ) with 0.5 M KC1 (Woodley et al., 1974) and t RN A was obtained also from rabbit reticulocytes as described by Bose et al. (1974).
Cell-free protein synthesis assay mixture The standard assay mixture contained in a total volume of 0.075 ml was c o mp o s ed of 18 mM Tris--HC1 (pll 7.5), 22 mM NaCl, 97 mM KC1. 3 mM Mg ÷*, 0.2 mM dithiothreitol (DTT), 1 mM ATP, 0.2 mM GTP, 4 mM PEP, 4 IU/ml pyruvate kinase, 67 pM each of 19 unlabeled amino acids, 6.66 pCi/ml 4,5-~H-leucine (67.6 Ci/mmole), 57 /ag/ml unfractionated tRNA. To this mixture 0.54 absorbance (260 nm) units $30 ribosomes, 0.23 absorbance (280 nm) units of rabbit reticulocyte ribosomal 0.5 M KC1 wash (factors) and various concentrations of m a m m a r y gland or liw~r RNA were added. Incubation was done at 30°C for 40 min.
Casein assay The p r o d u c t of the cell-free system was assayed for mouse casein by an appropriately modified immunoprecipitation procedure similar to that used for d etectio n of ovalbumin by Means et al. (1972). Mouse casein was prepared by Ca**-rennin precipitation of BALB/c mouse milk (Jeurgens et al., 1965) and rabbit antiserum to casein was induced by injecting mouse milk casein into rabbits according to the procedure described by Feldman et al. (1970) and Feldman (1974). The casein antiserum (adsorbed 1 : 1 with mouse serum) and tested by immunodiffusion in 0.5% agar buffered with 0.01 M neutral phosphate. Zones of precipitation were observed with mouse milk, Ca**-rennin precipitate (casein) of mouse milk extract of lactating m a m m a r y tissue. No reaction was observed with virgin m a m m a r y gland, liver e x t ract or mouse serum. The a n t i b o d y was also tested by an indirect radioi m m u n o p r e c i p i t a t i o n procedure. The casein antiserum was first incubated at 4°C with various antigens overnight, the ' : 51 labeled mouse casein was then added to the tubes and 2 hr later samples were assayed. Mouse casein was labeled with ~: sI according to the methods of Bale et al. (1966). Binding of 251 casein to the ant i body was negligible in tubes preincubated with milk and Ca*'-rennin precipitate but ~251 casein binding to the antiserum was very high in tubes with virgin m am m ar y gland and liver extracts, mouse serum or rennin as antigens, indicating a specific affinity of the antisera for
116
mouse casein. A cletailed a c c ount of this indirect immunoprecipitation procedure for mouse casein will be reported elsewhere. For detcction of casein in the p r o d u c t o f the cell-free system, 20 pl of anticasein serum was added in a small tube, followed by 20/al 5,% sodium d e o x y c h o l a t e (w/v), 12 ~1 .1% Triton X-100 (v/v), 30 pl reaction mixture, 1 pg mouse milk (skimmed) carrier plus buffer containing 1 mM NaPO4 (pH 7.5) and 15 mM NaCI to a final volume of 96 ~1. The mixture was allowed to stand for 30 min at r oom temperature, 1 ml of 'wash' buffer [10 mM NaPOa (pH 7.5), 150 mM NaCI, 35~ Triton X-100, and 10 mM unlabeled leucine] was then added and contents of the tubes were thoroughly mixed. The precipitate was collected on an 0.45 p Millipore filter, rinsed with 20 ml of the 'wash' buffer, dried and counted for radioactivity.
Urea polyacrylamide gel ele(.trophoresis Mouse milk carrier prepared by pll 4.6 precipitation of the milk was dissolved in imidazole (KC1, 0.15 M; NaPO4, 0.004; CaC12, 0.01 M; imidazole, 0.01 M; pH 6.8) bufh,r. 2 mg of mouse milk carrier was added to 370 pl of cell-free reaction mixture and the solution was made 0.01 M with respect to CaC12 and rennin was added to 0.85 mg/ml. The solution was then incubated at 37°C for 30 min; tile precipitate, collected by centrifugation, was washed in imidazole buffer, 5~ trichloroacetic acid (TCA), and dissolved m 7 M urea. 1 0 0 / l g samples per gel were then subjected to electrophoresis in 7V~ polyacrylamide gel in presence of 7 M urea (pH 8.9} as previously descrit)cd by Feldman et al. (1971).
SDS polyaco, lamide gel electrophoresis The mouse casein antibody and the antigen complex obtained by the m e t h o d described above (see casein assay) was dissolved in 50 gl of 1% sodium dodecyl sulfate (SDS) and 50 pl of 2 mg/ml skimmed mouse milk in 1% SDS was added to the solution. The mixture was then solubilized by heating at 70°C for 2 min. After addition of /3-mercaptoethanol (1% final c o n c e n t r a t i o n in 1% SDS) the solution was made 7 ~ with respect to sucrose and 0.02 mg/ml b r o m o p h e n o l blue and the samples were subjected to electrophoresis as described by Weber et al. (1969). Protein determinations were done by the methods of Lowry et al. (1951) using bovine casein as standard. All operations were carried out at 0--4°C e x c e p t where mentioned otherwise and radioactivity of the samples was determined by liquid scintillation s p e c t r o m e t r y in a Beckman LS 350 counter. RESULTS Data in Table I show the responsiveness of the cell-free protein synthesizing system derived f r om Ehrlich ascites t u m o r ribosomes and rabbit reticulo-
117 TABLE I Requirements for translation of exogenous mRNA in a cell-free protein synthesis system, prepared from Ehrlich's ascites tumor cells.
Exogenous RNA
Assay condition
cpm/0.1 ml reaction mixture
None None Liver Mammary gland None Liver Mammary gland
Reaction mixture + tRNA + S.~(~ + RNA + S~(j + RNA + S~o + F + S~) + RNA + F + S~o + RNA + F + S.~(,
390 5,600 15,200 18,000 44,200 101,600 119,440
Standard reaction mixture in 0.075 ml volume as described in Materials and Methods were used. RNAs were prepared from 6 l ( ) d a y lactating mice. The optimum concentrations of 6 u g liver RNA or 1 3 u g mammary gland RNA per 0.1 ml were used. Higher concentrations of RNA were inhihitory in this system. Aliquots of the reaction mixture were assayed for trichloroacetic acid insoluble :~tt-leucine radioactivity by the filter disc method of Woodley et al. (1974). Data represent typical values of 5 10determinations. F, rahhit reticulocyte ribosome salt (0.5 M KCI) wash factors, purified hy DEAE cellulose column chromatography according to Woodley et al. (197,1).
c y t e f a c t o r s . As m e a s u r e d b y "~H - l e u c i n e i n c o r p o r a t i o n i n t o an a c i d i n s o l u b l e m a t e r i a l , l i v e r o r m a m m a r y g l a n d m R N A d i r e c t e d p r o t e i n s y n t h e s i s in t h e cell-free system remained low when the rabbit reticulocyte factors were not i n c l u d e d in t h e r e a c t i o n m i x t u r e . T h e a c t i v i t y w a s h i g h e r w h e n t h e r e a c t i o n w a s c a r r i e d o u t w i t h a s c i t e s cell r i b o s o m e s a n d t h e r e t i c u l o c y t e f a c t o r b u t n o added mRNA. This increased activity presumably represents translation of the residual endogenous mRNA. Addition of the exogenous RNA from mouse liver or mammary gland produced more than 2-fold stimulation of ~*H - l e u c i n e i n c o r p o r a t i o n i n t o a n a c i d i n s o l u b l e m a t e r i a l . T h i s d e m o n s t r a t e s that the present cell-free protein synthesis system with Ehrlich ascites tumor c e l l r i b o s o m e s is d e p e n d e n t o n r a b b i t r e t i c u l o c y t e f a c t o r s a n d t h e s y s t e m is capable of effective translation of exogenous mRNA. T h e e x o g e n o u s m R N A d i r e c t e d p r o d u c t o f t h e c e l l - f r e e s y s t e m was t h e n a n a l y s e d f o r a s p e c i f i c m i l k - p r o t e i n , c a s e i n a n d t h e r e s u l t s a r e s h o w n in T a b l e I1. E v i d e n c e o f c a s e i n s y n t h e s i s w a s a b s e n t in ' t h e p r o d u c t o f t h e c e l l - f r e e s y s t e m w i t h n o a d d e d e x o g e n o u s m R N A a n d n o c a s e i n s y n t h e s i s was p r e s e n t in t h e r e a c t i o n p r o d u c t d i r e c t e d b y l i v e r m R N A . B u t R N A o f t h e l a c t a t i n g mammary gland directed the synthesis of a product which was precipitable by a specific antiserum to mouse milk casein. This observation indicates that t h e h e t e r o l o g o u s c e l l - f r e e p r o t e i n s y n t h e s i s s y s t e m d e r i v e d f r o m E h r l i c h asc i t e s t u m o r cell r i b o s o m e s a n d r a b b i t r e t i c u l o c y t e f a c t o r s is c a p a b l e o f f a i t h -
llH TABLE 1I Messenger activity of different RNA preparations on synthesis of casein in an Ehrlich ascites tumor cell ribosome protein synthesis system. Source of RNA .
.
.
.
.
.
.
.
.
None Liver Mammary gland
Casein epm/0.1 ml reaction mixture .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
0 0 I 1,750
IAwn" and mammary ~laud RNA were prepared from 6 10 day lactating mice. Casein cpm was determined by specific immunoprecipitation of the 31/.leucine labeled product of cell-free syslem and mouse casein antiserum. The l)recipitate was immobilized on a nilrocellulose fiher and counted for radioa('tivity as described in Materials and Methods. Normal rabbit serum (preimmune) background cpm(320- 1000)/0.1 ml reaction mixture were suhtracted from all casein counts, l)ala represent typical wdues of 3--6 determinations.
fully translating the m u r i n e m a m m a r y gland m R N A for casein, a specific milk p r o t e i n . T h e l a c t a t i n g m a m m a l 5 , gland and liver m R N A d i r e c t e d p r o d u c t m the cell-free s y s t e m was also a n a l y s e d by u r e a - 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 i s a n d t h e results are s h o w n in Fig. 1. C o n s i s t e n t with earlier r e p o r t s ( F e l d m a n et al., 1 9 7 1 ) m o u s e milk carrier casein (Ca++-rennin p r e c i p i t a t e ) a f t e r ureap 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 i s resolved into distinct a b s o r b a n c e b a n d s and the p r e s e n t p e a k s 3, 4, and 5 were c o r r e s p o n d i n g to similar regions o f the gel, p r e v i o u s l y identified as c o m p o n e n t s o f m o u s e milk casein. A high level o f r a d i o a c t i v i t y o f the m a m m a r y gland R N A d i r e c t e d p r o d u c t o f the cell-free s y s t e m was associated with the a b s o r b a n c e p e a k s 3, 4, and 5. Whereas the r a d i o a c t i v i t y of the liver R N A d i r e c t e d p r o d u c t did n o t s h o w a n y c o r r e s p o n d e n c e with these casein c o m p o n e n t s . These results again d e m o n strate the t r a n s l a t i o n o f the l a c t a t i n g m a m m a r y gland R N A into a specific milk p r o t e i n , casein in a h e t e r o l o g o u s cell-free p r o t e i n s y n t h e s i z i n g s y s t e m . As s h o w n in Fig. 2, casein, s y n t h e s i z e d in the cell-free s y s t e m , was t h e n c h a r a c t e r i z e d by e l e c t r o p h o r e s i s in p o l y a c r y l a m i d e gel c o n t a i n i n g s o d i u m d o d e c y l sulfate (SDS). A f t e r dissociating the antigen- a n t i b o d y c o m p l e x by h e a t the s o l u t i o n was s u b j e c t e d to e l e c t r o p h o r e s i s . The r a d i o a c t i v e p r o t e i n m i g r a t e d in the gel as a p r o n o u n c e d p e a k c o r r e s p o n d i n g to the light chain ( L-chain ) o f r a b b i t "),-globulin. F u r t h e r m o r e , similar e l e c t r o p h o r e s i s of m o u s e milk casein in parallel gels also resolved into s h a r p a b s o r b a n c e p e a k corres p o n d i n g to the p e a k o f the light chain. It is r e a s o n a b l e , t h e r e f o r e , to conclude t h a t the high level o f r a d i o a c t i v i t y associated with the light chain represenks a c o m p o n e n t of m o u s e milk casein actively s y n t h e s i z e d in the cell-free s y s t e m . T h e m o l e c u l a r weight o f the light chain of 7-globulin is 2 3 , 5 0 0 (Weber et al., 1969), and the m o l e c u l a r weight o f the b o v i n e ~
119
I
i
]
(
iI i
!
!/"
!
" I
ii • "
%;
fl
~.
""
.;.
Fig. 1. U r e a - p o l y a e r y l a m i d e gel e l e c t r o p h o r e s i s of the p r o d u c t of cell-free p r o t e i n s y n t h e sis s y s t e m . At the end o f the reaction, 2 m~ of m o u s e milk carrier was a d d e d to the r e a c t i o n m i x t u r e , casein was p r e c i p i t a t e d by Ca++-rennin. The p r e c i p i t a t e was then dissolved in 7 M urea, 100/.tg samples were layered on 71~ a e r y l a m i d e gel and duplicate gels were s u b j e c t e d to e l e c t r o p h o r e s i s (Davis, 1964) for 2..10 rain at 3 m A per gvl. One eel was stained with buffalo black, a b s o r b a n e e (400 tam) was measured in a Gitford gel scanner. The s e c o n d gel was s e c t i o n e d (1 ram) with a Miekel gel slicer. The sections were covered with 0.5 ml Nuclear Chicago Soluhilizer (NCS) in liquid scintillation vials. i n c u b a t e d at 50°C overnight and swelled s e c t i o n s were then c o u n t e d for radioactivity. Note t h a t 3 H-leucine radioactivity o f only the m a m m a r y gland RNA p r o d u c t c o r r e s p o n d s to a b s o r b a n c e p e a k s 3, 4, a n d 5 o f m o o s e c a s e i n . • • , M a m m a r y gland; • . . . . . A, Liver. Fig. 2. C h a r a c t e r i z a t i o n o f the p r o t e i n s s y n t h e s i z e d in the cell-free s y s t e m by p o l y a e r y l amide-SDS gel e l e e t r o p h o r e s i s . The anticasein p r e c i p i t a t e o f the cell-free p r o d u c t of m a m m a r y gland or liver RNA was collected and treated as descrihed in the e x p e r i m e n t p r o c e d u r e . 100t~g o f the material ( p r o t e i n s ) was layered on p o l y a c r y l a m i d e - S D S gel and s u b j e c t e d to e l e e t r o p h o r e s i s as described by Weber et al. (1969). Duplicate gels were run for each e x p e r i m e n t , one was stained with coomassie blue and a h s o r b a n e e (550 n m ) w a s m e a s u r e d in a Gilford gel scanner. The s e c o n d gel was s e c t i o n e d ( 1 ram) with a Mickel gel slicer, the s e c t i o n s were m o i s t e n e d in a liquid scintillation vial, 10 ml o f .1% NCS (in t o l u e n e - b a s e d scintillation fluid) was a d d e d and i n c u b a t e d at 37°C overnight. The swelled s e c t i o n s were t h e n c o u n t e d in a liquid scintillation system. Note the p r o n o u n c e d radioactivity o f m a m m a r y R N A p r o d u c t in the region o f the light chain o f the )'-globulin ahsorl)ance peak (arrow). Parallel p o l y a c r y l a m i d e - S D S gel e l e c t r o p h o r e s i s o f m o u s e milk casein also s h o w e d a sharp a b s o r b a n c e peak in the n,gion c o r r e s p o n d i n g to the light chain o f the y-globulin. S o m e radioactivity o f the m a m m a r y gland R N A p r o d u c t was also associated with the m i n o r peak o f milk p r o t e i n handing in the area o1' gel s e c t i o n s 19--22. Radioactivity o f the liw~r RNA p r o d u c t did n o t s h o w any association with the a h s o r h a n c e profih'. . . . . . . . . . . . . , M a m m a r y gland; o- o Liver.
( M e r c e i r e t al., 1 9 7 2 ) . T h u s , i n v i e w o f t h e c l o s e n e s s o f the molecular weight and the electrophoretic mobility pattern of the two proteins we assume that the bulk of the lactating m a m m a r y gland m R N A directed synthesis of casein in the present cell-free system represents a~ cornc a s e i n is 2 2 , 9 7 5
120 p o n e n t o f m o u s e casein, the major fraction of the milk protein. This interp r e t a t i o n is c o n s i s t e n t with the observation t h a t m R N A of lactating mammary gland o f the ewe directs d e t e c t a b l e synthesis o f only a s casein in a rabbit r e t i c u l o c y t e lysate protein synthesis system (Gaye et al., 1973). A m o d e s t a m o u n t of radioactivity in the p r o d u c t o f the lactating m a m m a r y gland RNA was also associated with a m i n o r peak of casein c o m p o n e n t in the S D S - p o l y a c r y l a m i d e gel banding in th(, region of gel sections 19- 22 as s h o w n in Fig. 2. I d e n t i f i c a t i o n o f this casein c o m p o n e n t synthesized in the cell-free s y s t e m is n o t clear at this time. DISCUSSION The p r e s e n t results d e m o n s t r a t e that messenger activity o f RNA isolated f r o m the lactating murine m a m m a r y gland is capable o f directing the synthesis o f a specific milk protein, casein in a cell-free p r o t e i n synthesis system. The results also revealed t h a t translation o f the m R N A for as casein in the ascites t u m o r cell r i b o s o m e s y s t e m does n o t require the presence of species specific factors and these observations are in a g r e e m e n t with similar results previously described in o t h e r e u c a r y o t i c systems ( G u r d o n et al., 1971; Mathews et al., 1971; Rhoads et al., 1972; Rosenfeld et al., 1972; Gaye et al., 1973). However, the present findings c o n s t i t u t e the first d e m o n s t r a t i o n t h a t m R N A for a specific milk p r o t e i n (or its c o m p o n e n t s ) can be faithfully translated in a cell-free p r o t e i n synthesis s y s t e m derived f r o m Ehrlich ascites t u m o r cell r i b o s o m e s and rabbit r e t i c u l o c y t e factors. It is not y e t k n o w n which f r a c t i o n o f the p o l y s o m a l RNA of the lactating m a m m a r y gland o f the mouse c o d e for the milk-protein and these studies are in progress. Preliminary results suggest t h a t p o l y s o m a l aggregates of ,1, 6, and 12 ribosomal units o f the lactating m a m m a r y gland are positive in casein m R N A activity. Multiple h o r m o n a l regulation o f the f u n c t i o n a l d i f f e r e n t i a t i o n o f m u r i n e mammary gland is well delineated (Nandi et al., 1961; T o p p e r , 1970; T u r k i n g t o n et al., 1973; Rivera, 1974; Wood et al., 1975), b u t the h o r m o n e or combination o f h o r m o n e s involved in the transcriptional regulation o f milk-protein m R N A y e t needs to be elucidated. It is n o w possible to simulate the biological cycle o f growth, lactation and involution o f the m a m m a r y p a r e n e h y m a in organ c u l t u r e o f the entire gland, by a p p r o p r i a t e l y regulating the h o r m o n a l s u p p l e m e n t a t i o n o f the serum-free s y n t h e t i c m e d i u m (Banerjee et al., 1974; Wood et al., 1975). E x t r a c t i o n and cell-free translation of fractions of RNA f r o m the gland cultivated in m e d i u m with various h o r m o n e s is c u r r e n t l y u n d e r w a y in o u r l a b o r a t o r y and the results should elucidate the mechanisms of i n d u c t i o n o f h o r m o n e m o d u l a t e d milk p r o t e i n m R N A in m a m m a r y differentiation. ACKNOWLEDGEMENTS Supported by a grant from the National Cancer Institute. We thank Prof~,ssor N.K.
121 Gupta of our Chemistry Department for advice and Dr. C. Woodley for help in developing the cell-free system, Mr. M. Loudon and Ms. Linda Washburn for technical assistance and Ms. Naomi Windhorst for seeretarial services. A preliminary report of these results was presented at the 14th Annual Meetings of the American Society of Cell Biology, San Diego, California, November 21 -23, 197,1.
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122 Weber, K. a n d M. O s b o r n : , l . Biol. Chem. 2.14,-1.t06 -t.112(1969). Wood, B.G., L.L. Washl)urn. A.S. M u k h e r j e e and M.II. Banerjee: ,I. l~;ndocr. 61 (in t)l'~,ss) ( 19T 5). Woodley, C.I,., Y.C. Chen and N.K. ( ; u p t a : I n : M e t h o d s of Enzymoh)gy>¢,ds. K. Mc>ldav,, and L. (h'¢~sman ( A c a d e m i c Pla,ss, New York ) p. 177 (19';'4).
