Vol. 65, No. 4, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
REPRESSION OF ARC mRNA SYNTHESIS
BY L - A R G I N I N E IN C E L L - F R E E
EXTRACTS OF ESCHERICHIA COLI.
P a l m e r " Roger's, T e d M . K a d e n , and M i c h a e l T o t h DepaPtment of Mtcr'obiology UntvePsity of Minnesota Medical School M i n n e a p o l i s , M i n n e s o t a 55455
Received June 26,1975 SUMMARY. C e t l - f r ` e e s y n t h e s i s o f s p e c i f i c messenger" R N A for" the ar-gE C B H gene cluster" o f E. c o l i w a s demonstr`ated w i t h cr`ude e x t r - a c t s , k d a r g D N A as t e m p l a t e , and by D N A - R N A h y b r ` t d i z a t i o n ol= 3 H - R N A to @80 d ar'g D N A . h - A r ' g t n t n e r'epr'essed the s y n t h e s i s o f a r ' g - m R N A a b o u t 60% in e x t r ' a c t s pr'epar'ed fr'om ar'g R + str'ains but had no e f f e c t in e x t r 'ac t s f r o m an ar'g R s t r a i n .
INTRODUCTION.
S y n t h e s i s o f the e i g h t e n z y m e s i n v o l v e d in the b i o -
s y n t h e s i s ol= aPgtnine tn E s c h e P t c h i a c o l t K1 2 ape n e g a t i v e l y c o n t P o l l e d by the pPoduct o f a s i n g l e PegulatoPy g e n e , ar,g R, w h i c h is a pPo t e in (1, 2). T h e coPepPessoP f o p PepPesston is eitheP ar-gtnine oP a d e P t v a t t v e o f a P g i n i n e such as a P g i n y l - t R N A (3, 4, 5).
HoweveP, s o m e e v i d e n c e s u g g e s t s that
a r ' g i n y l - t RN A is not the coPepPessoP (6, 7).
U s in g a celt--fPee s y s t e m ,
UPm, et a l . (2) d e m o n s t r - a te d that in v i t r ' o r'epPession o f N - e - a c e t y l - L o P n i t h i n a s e s y n t h e s i s PequtPed an a c t i v e aPg R + pPoduct.
But, since aPginine
and aPgtnyl t R N A ape e s s e n t i a l for" pr'otetn s y n t h e s i s in this s y s t e m , the coPepPessoP r-equir'ement w a s not s t u d i e d .
We d e s c P i b e hePe a cr`ude c e l t -
fr-ee s y s t e m f o p s t u d y o f a r ` g i n i n e - P e p P e s s t o n ol= the s y n t h e s i s of s p e c i f i c messenger" R N A f r o m the E. c o l t aPg E C B H Pegion ( a P g - m R N A ) .
While
pr`epar`ing this wor`k for` p u b l i c a t i o n , a r`epoPt by P a n n e k o e k , et a l . (8)
T h i s wor-k was suppor'ted by P u b l i c H e a l t h S e t ' v i c e gr,ant GM1 8953.
Copyright © 1975 by Academic Press, Inc. All rights o f reproduction in any form resenJed.
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a p p e a r e d d e s c r i b i n g in v i t r o a r g - m R N A
synthesis using purified
p o i y m e r a s e and J~80 d a r g E C B H as t e m p l a t e .
RNA
The cell-free arginine-
r e p r e s s i o n w e r e p o r t h e r e m a y be f u r t h e r r e s o l v e d u s i n g t h i s p u r i f i e d system,
MATERIALS AND METHODS. B a c t e r i a , P h a g e , and Phage DNA: E s c h e r t c h t a c o l t s t r a i n 201 W9 o b t a i n e d f r o m W. K . M a a s is a r g - t h r - l e u and .arg R-. In t h i s s t r a i n added a r g i n t n e does not r e p r e s s s y n t h e s i s o f the a r g i n i n e b i o s y n t h e t i c e n z y m e s . E. c o l t s t r a i n 0 6 0 0 & [ ' a r g E O B H - , p p c - ] ( f r o m N. G l a n s d o r f f ) c o n t a i n s d e l e t i o n c o v e r i n g the a r g E O B H and ppc r e g i o n s , and i s a r g R +, thus added a r g i n i n e r e p r e s s e s s y n t h e s i s o f the a r g i n t n e b i o s y n t h e t i c e n z y m e s , k d a r g No. 14 (a g i f t f r o m N. G t a n s d o r f f ) i s a d e f e c t i v e t r a n s d u c i n g phage c a r r y i n g a r g E O B H and p p c , and i s t h e r m o i n d u c i b l e ( 0 1 8 5 7 ) and [ y s i s d e f e c t i v e ( S 7 ) . I t ts g r o w n w i t h a h e l p e r X . ~80 d a r g a l s o c a r r i e d a r g E O B H and ppc and w a s i s o l a t e d by B . K o n r a d . I t is a l s o g r o w n w i t h a h e l p e r j~80. ~80h X (a g i f t f r o m R . P r e s s ) is a h y b r i d ~phage c o n t a t n i n g the X O1857 r e g i o n and is t h e r m o i n d u c t b l e . Large batches of X+, ~80 +, and ~80 d a r g phages w e r e p r e p a r e d by i n d u c i n g l y s o g e n s w i t h u l t r a v i o l e t t i g h t (9) and k d a r g and ~80h X w e r e i n d u c e d by r a i s i n g the t e m p e r a t u r e f o r 25 m t n f r o m 82 ° to 41 o. A f t e r i n d u c t i o n c u l t u r e s w e r e i n c u b a t e d f o r 8 h r s . at 3 7 ° 0 . X d a r g phage was e x t r a c t e d by c h l o r o f o r m treatment from induced ceils concentrated by centrifugatton Phages X + ~80 +, ~80 d a r g , and ~80h X w e r e p r e c i p i t a t e d w i t h p o l y e t h y l e n e g l y c o l (10). A l l phages w e r e p u r i f i e d and t r a n s d u c i n g phages w e r e s e p a r a t e d f r o m h e l p e r phages b y two i s o p y c n t c c e n t r i f u g a t t o n s i n 0 S 0 l . Phage D N A w a s e x t r a c t e d as d e s c r i b e d p r e v i o u s l y (11 ) and d i a l y z e d 1 8 h r s at 4 ° 0 a g a t n s t b u f f e r c o n t a i n i n g : 1 0 m M T r i s - a c e t a t e , pH 7 . 8 ; 50 m M K O I ; and 1 m M E D T A and s t o r e d at 4 ° 0 o v e r c h l o r o f o r m . In v i t r o t r a n s c r i p t i o n s y s t e m : S t r a i n s 0 6 0 0 '~ [ a r g E O B H , ppc'] and 201W9 w e r e g r o w n tn 5 l i t e r b a t c h e s at 3 5 ° 0 w i t h v i g o r o u s a e r a t i o n to 2 x 108 c e l l s / m l i n m e d i u m c o n t a i n i n g p e r l i t e r : 10gin n u t r i e n t b r o t h ; 5 gm a a c t o peptone ( D t f c o ) ; 1 g m g l u c o s e ; 10 mg t h i a m i n e ; and 20 mg t h y m i n e . C e l l f r e e e x t r a c t s w e r e p r e p a r e d f r o m w a s h e d c e l t s e s s e n t i a l l y by the m e t h o d o f M i l l e r (1 2) and s t o r e d at - 8 0 ° 0 f o l l o w i n g d i a l y s i s . For RNA synthesis, r e a c t i o n m i x t u r e s o f 0.1 m l c o n t a i n e d : 50 m M p o t a s s i u m acetate; 25 m M a m m o n i u m acetate; 40 m M T r t s - a c e t a t e , pH 8 . 2 ; 10 m a m a g n e s i u m acetate; 1 .4 m M d t t h t o t h r e i t o l ; 1 m M E D T A ; 0 . 4 m M each o f A T P , O T P , and G T P ; 0 . 2 m M and 20 I• Ot 3 H - U T P ; 5 t~g o f X o r k d a r g E)NA; and 660 Fig e x t r a c t p r o t e i n . T o s o m e tubes L - a r g t n i n e w a s added at the c o n c e n t r a t i o n s s h o w n . R e a c t i o n m i x t u r e s w e r e u s u a l l y p r e i n c u b a t e d 1 0 m t n at 2 0 ° C w i t h o u t r i b o n u c t e o s t d e t r i p h o s p h a t e s and t h e n the t r i p h o s p h a t e s w e r e added and the r e a c t i o n m i x t u r e s w e r e i n c u b a t e d f o r 1 5 rain a t 3 7 ° 0 . R e a c t i o n s w e r e stopped b y i c i n g ; 800 I~g R N A o f E . c o l t s t r a i n 0 6 0 0 /~[arg E O B H , p p c ] and 10 Fg DNase ( R N a s e f r e e ) w e r e added; and, a f t e r 15 m t n on i c e , R N A w a s e x t r a c t e d e s s e n t i a l l y as d e s c r i b e d b y O r o m b r u g g h e , et a l . (13). D N A - R N A h y b r i d i z a t i o n : A l k a l i d e n a t u r e d phage D N A w a s f i x e d to n i t r o c e l l u l o s e m e m b r a n e f i l t e r s at 1 0 Fig per- d i s k as p r e v i o u s l y d e s c r i b e d (11 ).
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H y b P i d i z a t t o n Peacttons wePe caPPied out in 1 .1 m l 2 x S S O * f o p 18 h r a t 6 7 ° 0 and d i s k s wePe w a s h e d , tPeated w i t h RNase~ and counted as descPibed p P e v i o u s l y (11 ). RESULTS.
In p P e l t m i n a P y e x p e P i m e n t s , cPude extPacts o f E. c o l t 0 6 0 0
tl [-aPg E O B H , p.pc] wePe found to incoPpoPate 3 0 0 - 4 0 0 p m o l e s of: 3 H - U T P into a c i d p P e c i p i t a b l e R N a s e s e n s i t i v e m a t e P i a l ( R N A) pePiod undeP the Peaction c o n d i t i o n s descPtbed.
oveP a 1 0 to 15 r a i n
Ad d e d phage D N A (5 tzg pep
0.1 m l ) enhanced i n c o P p o P a t i o n up to 7 0 0 - 9 0 0 p m o l e s .
Th e Pe was l i t t l e oP
no e f f e c t o f added L - a P g i n t n e oP t R N A duping a 10 m t n p P e i n c u b a t i o n pePiod at 2 0 ° 0 eitheP on the t i m e couPse oP y i e l d o f 3 H - R N A f o P m e d . In oPdeP to d e t e c t R N A tPanscPibed s p e c i f i c a l l y f:Pom E. c o t i aPg genes on the k d aPg t e m p t a t e ( T a b l e 1), the 3 H - R N A ex t Pac t e d fPom Peaction m t x t u P e s was h y b P i d i z e d w i t h ~80 and ~80 d aPg D N A .
As PepoPted pPe-
v i o u s l y (1 4) thePe was l i t t t e c P o s s - P e a c t i v i t y b e t w e e n k and ~80 tPanscPipts, and a b o u t 1.5% o f the i n p u t 3 H - R N A was tPanscPibed fPom the aPg E C B H ppc Pegion, ( T a b l e 1).
S i n c e the e x t r a c t was fPom an aPgR + s t r a i n , the e f f e c t
o f added h - a P g i n i n e on a r ' g - m R N A t P a n s c P i p t i o n w as t e s t e d .
( T a b l e s 1 and 2).
T h e data s h o w t h a t 4 m M h - a P g i n i n e Peduced the a m o u n t o f a P g - m R N A to a b o u t 40% o f that p r o d u c e d w i t h o u t a P g i n i n e .
H y b P i d i z a t i o n o f the s a m e
3 H - R N A pPepaPations w i t h ~80 h k D N A showed that the t P a n s c P i p t i o n o f X D N A in the C I Pegion occuPPed and was not s e n s i t i v e to aPgtntne PepPession. V a P i o u s c o n c e n t r a t i o n s o f aPgtnine wePe tested ( F i g . 1) and m a x i m u m PepPession was e l i c i t e d by 0 . 5 m M L - a P g i n i n e in t h i s t e s t s y s t e m .
In
a d d i t i o n to D - a P g i n t n e , otheP L - a m i n o a c t d s at 5 r a m such as l y s t n e , o P n i t h i n e , cttPultine~ and c a n a v a n i n e did n o t y i e l d any r e p P e s s i o n o f a P g - m
* S S O is 0 . 1 5 NaOl - - 0 . 0 1 5 M s o d i u m c i t P a t e .
1286
Vol. 65, No. 4, 1975
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.Z U)
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BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
REPRESSION OF ARC mRNA SYNTHESIS
BY L - A R G I N I N E IN C E L L - F R E E
EXTRACTS OF ESCHERICHIA COLI.
P a l m e r " Roger's, T e d M . K a d e n , and M i c h a e l T o t h DepaPtment of Mtcr'obiology UntvePsity of Minnesota Medical School M i n n e a p o l i s , M i n n e s o t a 55455
Received June 26,1975 SUMMARY. C e t l - f r ` e e s y n t h e s i s o f s p e c i f i c messenger" R N A for" the ar-gE C B H gene cluster" o f E. c o l i w a s demonstr`ated w i t h cr`ude e x t r - a c t s , k d a r g D N A as t e m p l a t e , and by D N A - R N A h y b r ` t d i z a t i o n ol= 3 H - R N A to @80 d ar'g D N A . h - A r ' g t n t n e r'epr'essed the s y n t h e s i s o f a r ' g - m R N A a b o u t 60% in e x t r ' a c t s pr'epar'ed fr'om ar'g R + str'ains but had no e f f e c t in e x t r 'ac t s f r o m an ar'g R s t r a i n .
INTRODUCTION.
S y n t h e s i s o f the e i g h t e n z y m e s i n v o l v e d in the b i o -
s y n t h e s i s ol= aPgtnine tn E s c h e P t c h i a c o l t K1 2 ape n e g a t i v e l y c o n t P o l l e d by the pPoduct o f a s i n g l e PegulatoPy g e n e , ar,g R, w h i c h is a pPo t e in (1, 2). T h e coPepPessoP f o p PepPesston is eitheP ar-gtnine oP a d e P t v a t t v e o f a P g i n i n e such as a P g i n y l - t R N A (3, 4, 5).
HoweveP, s o m e e v i d e n c e s u g g e s t s that
a r ' g i n y l - t RN A is not the coPepPessoP (6, 7).
U s in g a celt--fPee s y s t e m ,
UPm, et a l . (2) d e m o n s t r - a te d that in v i t r ' o r'epPession o f N - e - a c e t y l - L o P n i t h i n a s e s y n t h e s i s PequtPed an a c t i v e aPg R + pPoduct.
But, since aPginine
and aPgtnyl t R N A ape e s s e n t i a l for" pr'otetn s y n t h e s i s in this s y s t e m , the coPepPessoP r-equir'ement w a s not s t u d i e d .
We d e s c P i b e hePe a cr`ude c e l t -
fr-ee s y s t e m f o p s t u d y o f a r ` g i n i n e - P e p P e s s t o n ol= the s y n t h e s i s of s p e c i f i c messenger" R N A f r o m the E. c o l t aPg E C B H Pegion ( a P g - m R N A ) .
While
pr`epar`ing this wor`k for` p u b l i c a t i o n , a r`epoPt by P a n n e k o e k , et a l . (8)
T h i s wor-k was suppor'ted by P u b l i c H e a l t h S e t ' v i c e gr,ant GM1 8953.
Copyright © 1975 by Academic Press, Inc. All rights o f reproduction in any form resenJed.
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a p p e a r e d d e s c r i b i n g in v i t r o a r g - m R N A
synthesis using purified
p o i y m e r a s e and J~80 d a r g E C B H as t e m p l a t e .
RNA
The cell-free arginine-
r e p r e s s i o n w e r e p o r t h e r e m a y be f u r t h e r r e s o l v e d u s i n g t h i s p u r i f i e d system,
MATERIALS AND METHODS. B a c t e r i a , P h a g e , and Phage DNA: E s c h e r t c h t a c o l t s t r a i n 201 W9 o b t a i n e d f r o m W. K . M a a s is a r g - t h r - l e u and .arg R-. In t h i s s t r a i n added a r g i n t n e does not r e p r e s s s y n t h e s i s o f the a r g i n i n e b i o s y n t h e t i c e n z y m e s . E. c o l t s t r a i n 0 6 0 0 & [ ' a r g E O B H - , p p c - ] ( f r o m N. G l a n s d o r f f ) c o n t a i n s d e l e t i o n c o v e r i n g the a r g E O B H and ppc r e g i o n s , and i s a r g R +, thus added a r g i n i n e r e p r e s s e s s y n t h e s i s o f the a r g i n t n e b i o s y n t h e t i c e n z y m e s , k d a r g No. 14 (a g i f t f r o m N. G t a n s d o r f f ) i s a d e f e c t i v e t r a n s d u c i n g phage c a r r y i n g a r g E O B H and p p c , and i s t h e r m o i n d u c i b l e ( 0 1 8 5 7 ) and [ y s i s d e f e c t i v e ( S 7 ) . I t ts g r o w n w i t h a h e l p e r X . ~80 d a r g a l s o c a r r i e d a r g E O B H and ppc and w a s i s o l a t e d by B . K o n r a d . I t is a l s o g r o w n w i t h a h e l p e r j~80. ~80h X (a g i f t f r o m R . P r e s s ) is a h y b r i d ~phage c o n t a t n i n g the X O1857 r e g i o n and is t h e r m o i n d u c t b l e . Large batches of X+, ~80 +, and ~80 d a r g phages w e r e p r e p a r e d by i n d u c i n g l y s o g e n s w i t h u l t r a v i o l e t t i g h t (9) and k d a r g and ~80h X w e r e i n d u c e d by r a i s i n g the t e m p e r a t u r e f o r 25 m t n f r o m 82 ° to 41 o. A f t e r i n d u c t i o n c u l t u r e s w e r e i n c u b a t e d f o r 8 h r s . at 3 7 ° 0 . X d a r g phage was e x t r a c t e d by c h l o r o f o r m treatment from induced ceils concentrated by centrifugatton Phages X + ~80 +, ~80 d a r g , and ~80h X w e r e p r e c i p i t a t e d w i t h p o l y e t h y l e n e g l y c o l (10). A l l phages w e r e p u r i f i e d and t r a n s d u c i n g phages w e r e s e p a r a t e d f r o m h e l p e r phages b y two i s o p y c n t c c e n t r i f u g a t t o n s i n 0 S 0 l . Phage D N A w a s e x t r a c t e d as d e s c r i b e d p r e v i o u s l y (11 ) and d i a l y z e d 1 8 h r s at 4 ° 0 a g a t n s t b u f f e r c o n t a i n i n g : 1 0 m M T r i s - a c e t a t e , pH 7 . 8 ; 50 m M K O I ; and 1 m M E D T A and s t o r e d at 4 ° 0 o v e r c h l o r o f o r m . In v i t r o t r a n s c r i p t i o n s y s t e m : S t r a i n s 0 6 0 0 '~ [ a r g E O B H , ppc'] and 201W9 w e r e g r o w n tn 5 l i t e r b a t c h e s at 3 5 ° 0 w i t h v i g o r o u s a e r a t i o n to 2 x 108 c e l l s / m l i n m e d i u m c o n t a i n i n g p e r l i t e r : 10gin n u t r i e n t b r o t h ; 5 gm a a c t o peptone ( D t f c o ) ; 1 g m g l u c o s e ; 10 mg t h i a m i n e ; and 20 mg t h y m i n e . C e l l f r e e e x t r a c t s w e r e p r e p a r e d f r o m w a s h e d c e l t s e s s e n t i a l l y by the m e t h o d o f M i l l e r (1 2) and s t o r e d at - 8 0 ° 0 f o l l o w i n g d i a l y s i s . For RNA synthesis, r e a c t i o n m i x t u r e s o f 0.1 m l c o n t a i n e d : 50 m M p o t a s s i u m acetate; 25 m M a m m o n i u m acetate; 40 m M T r t s - a c e t a t e , pH 8 . 2 ; 10 m a m a g n e s i u m acetate; 1 .4 m M d t t h t o t h r e i t o l ; 1 m M E D T A ; 0 . 4 m M each o f A T P , O T P , and G T P ; 0 . 2 m M and 20 I• Ot 3 H - U T P ; 5 t~g o f X o r k d a r g E)NA; and 660 Fig e x t r a c t p r o t e i n . T o s o m e tubes L - a r g t n i n e w a s added at the c o n c e n t r a t i o n s s h o w n . R e a c t i o n m i x t u r e s w e r e u s u a l l y p r e i n c u b a t e d 1 0 m t n at 2 0 ° C w i t h o u t r i b o n u c t e o s t d e t r i p h o s p h a t e s and t h e n the t r i p h o s p h a t e s w e r e added and the r e a c t i o n m i x t u r e s w e r e i n c u b a t e d f o r 1 5 rain a t 3 7 ° 0 . R e a c t i o n s w e r e stopped b y i c i n g ; 800 I~g R N A o f E . c o l t s t r a i n 0 6 0 0 /~[arg E O B H , p p c ] and 10 Fg DNase ( R N a s e f r e e ) w e r e added; and, a f t e r 15 m t n on i c e , R N A w a s e x t r a c t e d e s s e n t i a l l y as d e s c r i b e d b y O r o m b r u g g h e , et a l . (13). D N A - R N A h y b r i d i z a t i o n : A l k a l i d e n a t u r e d phage D N A w a s f i x e d to n i t r o c e l l u l o s e m e m b r a n e f i l t e r s at 1 0 Fig per- d i s k as p r e v i o u s l y d e s c r i b e d (11 ).
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BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
H y b P i d i z a t t o n Peacttons wePe caPPied out in 1 .1 m l 2 x S S O * f o p 18 h r a t 6 7 ° 0 and d i s k s wePe w a s h e d , tPeated w i t h RNase~ and counted as descPibed p P e v i o u s l y (11 ). RESULTS.
In p P e l t m i n a P y e x p e P i m e n t s , cPude extPacts o f E. c o l t 0 6 0 0
tl [-aPg E O B H , p.pc] wePe found to incoPpoPate 3 0 0 - 4 0 0 p m o l e s of: 3 H - U T P into a c i d p P e c i p i t a b l e R N a s e s e n s i t i v e m a t e P i a l ( R N A) pePiod undeP the Peaction c o n d i t i o n s descPtbed.
oveP a 1 0 to 15 r a i n
Ad d e d phage D N A (5 tzg pep
0.1 m l ) enhanced i n c o P p o P a t i o n up to 7 0 0 - 9 0 0 p m o l e s .
Th e Pe was l i t t l e oP
no e f f e c t o f added L - a P g i n t n e oP t R N A duping a 10 m t n p P e i n c u b a t i o n pePiod at 2 0 ° 0 eitheP on the t i m e couPse oP y i e l d o f 3 H - R N A f o P m e d . In oPdeP to d e t e c t R N A tPanscPibed s p e c i f i c a l l y f:Pom E. c o t i aPg genes on the k d aPg t e m p t a t e ( T a b l e 1), the 3 H - R N A ex t Pac t e d fPom Peaction m t x t u P e s was h y b P i d i z e d w i t h ~80 and ~80 d aPg D N A .
As PepoPted pPe-
v i o u s l y (1 4) thePe was l i t t t e c P o s s - P e a c t i v i t y b e t w e e n k and ~80 tPanscPipts, and a b o u t 1.5% o f the i n p u t 3 H - R N A was tPanscPibed fPom the aPg E C B H ppc Pegion, ( T a b l e 1).
S i n c e the e x t r a c t was fPom an aPgR + s t r a i n , the e f f e c t
o f added h - a P g i n i n e on a r ' g - m R N A t P a n s c P i p t i o n w as t e s t e d .
( T a b l e s 1 and 2).
T h e data s h o w t h a t 4 m M h - a P g i n i n e Peduced the a m o u n t o f a P g - m R N A to a b o u t 40% o f that p r o d u c e d w i t h o u t a P g i n i n e .
H y b P i d i z a t i o n o f the s a m e
3 H - R N A pPepaPations w i t h ~80 h k D N A showed that the t P a n s c P i p t i o n o f X D N A in the C I Pegion occuPPed and was not s e n s i t i v e to aPgtntne PepPession. V a P i o u s c o n c e n t r a t i o n s o f aPgtnine wePe tested ( F i g . 1) and m a x i m u m PepPession was e l i c i t e d by 0 . 5 m M L - a P g i n i n e in t h i s t e s t s y s t e m .
In
a d d i t i o n to D - a P g i n t n e , otheP L - a m i n o a c t d s at 5 r a m such as l y s t n e , o P n i t h i n e , cttPultine~ and c a n a v a n i n e did n o t y i e l d any r e p P e s s i o n o f a P g - m
* S S O is 0 . 1 5 NaOl - - 0 . 0 1 5 M s o d i u m c i t P a t e .
1286
Vol. 65, No. 4, 1975
BIOCHEMICALAND BIOPHYSICAL RESEARCH COMMUNICATIONS
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