M U T A T O R A N D UV-RESISTANCE P L A S M I D S FROM I N C O M P A T I B I L I T Y G R O U P S OTHER THAN N R . J . P i n n e y , Microbiology S e c t i o n , Department of P h a r m a c e u t i c s , The School of Pharmacy, U n i v e r s i t y o f London, Brunswick Square, London, WClN 1 A X
The s e n s i t i v i t y of t h e Ames t e s t f o r chemical mutagens h a s been i n c r e a s e d by t r a n s f e r of t h e N i n c o m p a t i b i l i t y group plasmid pKMlOl i n t o t h e t e s t e r s t r a i n s (McCannand o t h e r s , 1 9 7 5 ) . Some o t h e r N group p l a s m i d s i n c r e a s e UV o r chemicalm u t a g e n e s i s i n Salmonella o r E s c h e r i c h i a c o l i s t r a i n s and a l s o c o n f e r W resist a n c e on t h e i r h o s t s (Drabble and S t o c k e r , 1968; MacPhee, 1973; T r i b e and Pinney, 1 9 7 7 ) . To d e t e r m i n e how u n i v e r s a l t h e plasmid mediated m u t a t o r e f f e c t i s , and i n a n a t t e m p t t o f i n d p l a s m i d s w i t h even g r e a t e r m u t a t o r a c t i v i t i e s t h a n pKMlOl, p l a s m i d s from 19 i n c o m p a t i b i l i t y groups have been i n v e s t i g a t e d f o r m u t a t o r and UV-protecting a c t i v i t y . The a b i l i t y of a plasmid t o c a u s e m u t a t i o n w a s t e s t e d by e x p o s i n g plasmidc o n t a i n i n g (P+) s t r a i n s o f E . c o l i 343/113 l y s a r g . t o 40 J / m 2 of UV l i g h t as d e s c r i b e d by T r i b e and Pinney (19771, and comparing t h e f r e q u e n c y of r e v e r s i o n t o l y s i n e independence w i t h t h a t o b t a i n e d i n t h e p l a s m i d - l e s s (P-) s t r a i n . W p r o t e c t i o n was t e s t e d by comparing s u r v i v a l of washed o v e r n i g h t b r o t h c u l t u r e s of P+ and P- s t r a i n s a f t e r e x p o s u r e t o a UV d o s e of 240 J / m 2 . P l a s m i d s from twelve i n c o m p a t i b i l i t y groups showed no s i g n i f i c a n t i n c r e a s e i n These were: F I , F I I , A , C , H 1 , H2, 12, P, mutagenic a c t i v i t y o r W r e s i s t a n c e . S , T , W and X . Plasmids from seven groups s i g n i f i c a n t l y i n c r e a s e d post-UV r e v e r s i o n t o l y s i n e p r o t o t r o p h y i n E . c o l i s t r a i n 343/113 and a l s o d e c r e a s e d t h e W s e n s i t i v i t - y of t h i s s t r a i n . These were: FIV, M , B , 11, I y , N and 0. G r e a t e s t i n c r e a s e i n m u t a t i o n f r e q u e n c y w a s found w i t h plasmid pKMlOl, which f u l l y j u s t i f i e s i t s u s e i n t h e Ames t e s t e r s t r a i n s . The N g r o u p p l a s m i d R390 was e x c e p t i o n a l i n t h a t a l t h o u g h it i n c r e a s e d m u t a t i o n f r e q u e n c y , it e x e r t e d no Wp r o t e c t i n g a c t i v i t y on i t s h o s t . A s e r i e s o f d e l e t i o n m u t a n t s was o b t a i n e d and t e s t e d f o r a l l k n o w n p h e n o t y p i c t r a i t s o f R390. The f o l l o w i n g o r d e r of l i n k a g e was determined: -Cm-Tc-Tra- (Rep-Ap) -Mut- (Mod-Res) - (Sm-Su) T h i s a g r e e s w e l l w i t h t h e R3YO gene map of C o e t z e e , D a t t a and Hedges (1972) and e x t e n d s it t o i n c l u d e t h e p o s i t i o n o f t h e m u t a t o r (Mut) gene. A m p i c i l l i n r e s i s t a n c e (Ap) i s c a r r i e d on t r a n s p o s o n A (TnA) i n R390 ( H e f f r o n and o t h e r s , 19751, MacPhee (1974) h a s s u g g e s t e d t h a t and i t s p r o x i m i t y t o Mut may be s i g n i f i c a n t . W p r o t e c t i o n c o n f e r r e d by a n o t h e r N group plasmid R-Utrecht i s due t o a plasmidI f t h e m u t a t o r e f f e c t of R390 i s a l s o due t o plasmidcoded DNA polymerase. mediated e r r o r - p r o n e DNA polymerase a c t i v i t y , t h e n t h e i n s e r t i o n o f TnA i n t o Mut may have d e s t r o y e d t h e UV r e p a i r a c t i v i t y of t h e M u t gene p r o d u c t w i t h o u t a b o l i s h i n g i t s mutagenic f u n c t i o n . I thank D r s R.W. Hedges and H . R . Smith f o r p l a s m i d s , and am most g r a t e f u l t o Joyce Dunachie f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e .
C o e t z e e , J . N . , D a t t a , N. and Hedges, R.W. ( 1 9 7 2 ) , J . g e n . M i c r o b i o l . , 72, 543-552. ( 1 9 6 8 ) . J . g e n . M i c r o b i o l . , 53, 109-123. D r a b b l e , W.T. and S t o c k e r , B . A . D . H e f f r o n , F . , S u b l e t t , R . and o t h e r s ( 1 9 7 5 ) . J . B a c t e r i o l . , 1 2 2 , 250-256. MacPhee, D.G. (1973). Mut.Res., 18, 367-370. MacPhee, D . G . ( i 9 7 4 ) . N a t u r e . Lond.,251, 432-434. McCann, J . , S p i n g a r n , N . E . and o t h e r s (1975). Proc . n a t A c a d Sci. USA., 72,979-987. T r i b e , M.J. and Pinney, R . J . ( 1 9 7 7 ) . J.Pharm. Pharmac., 29, 68P.
The s e n s i t i v i t y of t h e Ames t e s t f o r chemical mutagens h a s been i n c r e a s e d by t r a n s f e r of t h e N i n c o m p a t i b i l i t y group plasmid pKMlOl i n t o t h e t e s t e r s t r a i n s (McCannand o t h e r s , 1 9 7 5 ) . Some o t h e r N group p l a s m i d s i n c r e a s e UV o r chemicalm u t a g e n e s i s i n Salmonella o r E s c h e r i c h i a c o l i s t r a i n s and a l s o c o n f e r W resist a n c e on t h e i r h o s t s (Drabble and S t o c k e r , 1968; MacPhee, 1973; T r i b e and Pinney, 1 9 7 7 ) . To d e t e r m i n e how u n i v e r s a l t h e plasmid mediated m u t a t o r e f f e c t i s , and i n a n a t t e m p t t o f i n d p l a s m i d s w i t h even g r e a t e r m u t a t o r a c t i v i t i e s t h a n pKMlOl, p l a s m i d s from 19 i n c o m p a t i b i l i t y groups have been i n v e s t i g a t e d f o r m u t a t o r and UV-protecting a c t i v i t y . The a b i l i t y of a plasmid t o c a u s e m u t a t i o n w a s t e s t e d by e x p o s i n g plasmidc o n t a i n i n g (P+) s t r a i n s o f E . c o l i 343/113 l y s a r g . t o 40 J / m 2 of UV l i g h t as d e s c r i b e d by T r i b e and Pinney (19771, and comparing t h e f r e q u e n c y of r e v e r s i o n t o l y s i n e independence w i t h t h a t o b t a i n e d i n t h e p l a s m i d - l e s s (P-) s t r a i n . W p r o t e c t i o n was t e s t e d by comparing s u r v i v a l of washed o v e r n i g h t b r o t h c u l t u r e s of P+ and P- s t r a i n s a f t e r e x p o s u r e t o a UV d o s e of 240 J / m 2 . P l a s m i d s from twelve i n c o m p a t i b i l i t y groups showed no s i g n i f i c a n t i n c r e a s e i n These were: F I , F I I , A , C , H 1 , H2, 12, P, mutagenic a c t i v i t y o r W r e s i s t a n c e . S , T , W and X . Plasmids from seven groups s i g n i f i c a n t l y i n c r e a s e d post-UV r e v e r s i o n t o l y s i n e p r o t o t r o p h y i n E . c o l i s t r a i n 343/113 and a l s o d e c r e a s e d t h e W s e n s i t i v i t - y of t h i s s t r a i n . These were: FIV, M , B , 11, I y , N and 0. G r e a t e s t i n c r e a s e i n m u t a t i o n f r e q u e n c y w a s found w i t h plasmid pKMlOl, which f u l l y j u s t i f i e s i t s u s e i n t h e Ames t e s t e r s t r a i n s . The N g r o u p p l a s m i d R390 was e x c e p t i o n a l i n t h a t a l t h o u g h it i n c r e a s e d m u t a t i o n f r e q u e n c y , it e x e r t e d no Wp r o t e c t i n g a c t i v i t y on i t s h o s t . A s e r i e s o f d e l e t i o n m u t a n t s was o b t a i n e d and t e s t e d f o r a l l k n o w n p h e n o t y p i c t r a i t s o f R390. The f o l l o w i n g o r d e r of l i n k a g e was determined: -Cm-Tc-Tra- (Rep-Ap) -Mut- (Mod-Res) - (Sm-Su) T h i s a g r e e s w e l l w i t h t h e R3YO gene map of C o e t z e e , D a t t a and Hedges (1972) and e x t e n d s it t o i n c l u d e t h e p o s i t i o n o f t h e m u t a t o r (Mut) gene. A m p i c i l l i n r e s i s t a n c e (Ap) i s c a r r i e d on t r a n s p o s o n A (TnA) i n R390 ( H e f f r o n and o t h e r s , 19751, MacPhee (1974) h a s s u g g e s t e d t h a t and i t s p r o x i m i t y t o Mut may be s i g n i f i c a n t . W p r o t e c t i o n c o n f e r r e d by a n o t h e r N group plasmid R-Utrecht i s due t o a plasmidI f t h e m u t a t o r e f f e c t of R390 i s a l s o due t o plasmidcoded DNA polymerase. mediated e r r o r - p r o n e DNA polymerase a c t i v i t y , t h e n t h e i n s e r t i o n o f TnA i n t o Mut may have d e s t r o y e d t h e UV r e p a i r a c t i v i t y of t h e M u t gene p r o d u c t w i t h o u t a b o l i s h i n g i t s mutagenic f u n c t i o n . I thank D r s R.W. Hedges and H . R . Smith f o r p l a s m i d s , and am most g r a t e f u l t o Joyce Dunachie f o r e x c e l l e n t t e c h n i c a l a s s i s t a n c e .
C o e t z e e , J . N . , D a t t a , N. and Hedges, R.W. ( 1 9 7 2 ) , J . g e n . M i c r o b i o l . , 72, 543-552. ( 1 9 6 8 ) . J . g e n . M i c r o b i o l . , 53, 109-123. D r a b b l e , W.T. and S t o c k e r , B . A . D . H e f f r o n , F . , S u b l e t t , R . and o t h e r s ( 1 9 7 5 ) . J . B a c t e r i o l . , 1 2 2 , 250-256. MacPhee, D.G. (1973). Mut.Res., 18, 367-370. MacPhee, D . G . ( i 9 7 4 ) . N a t u r e . Lond.,251, 432-434. McCann, J . , S p i n g a r n , N . E . and o t h e r s (1975). Proc . n a t A c a d Sci. USA., 72,979-987. T r i b e , M.J. and Pinney, R . J . ( 1 9 7 7 ) . J.Pharm. Pharmac., 29, 68P.
