Nucleic Acids Research Application of an immunoprecipitation procedure to the study of SV40 tumor antigen interaction with mouse genomic DNA sequences
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چکیده
S i m i a n V i r u s 4 0 ( S V 4 0 ) l a r g e T a n t i g e n i s a D N A b i n d i n g p r o t e i n w i t h h i g h a f f i n i t y f o r s e g m e n t s o f t h e v i r a l g e n o m e . T o f i n d o u t w h e t h e r T a n t i g e n a l s o b i n d s t o s e q u e n c e s o f g e n o m i c c e l l u l a r D N A w e m i x e d T a n t i g e n a n d S A U 3 A r e s t r i c t e d m o u s e D N A u n d e r s t r i n g e n t D N A b i n d i n g c o n d i t i o n s . R e s u l t i n g p r o t e i n D N A c o m p l e x e s w e r e i m m u n o p r e c i p i t a t e d u s i n g T a n t i g e n s p e c i f i c m o n o c l o n a l o r p o l y c l o n a l a n t i b o d i e s . T h e D N A f r a g m e n t s in t h e i m m u n o p r e c i p I t a t e s w e r e c l o n e d in p l a s m i d v e c t o r s . F o u r p l a s m i d c l o n e s w e r e s e l e c t e d f o r a d e t a i l e d i n v e s t i g a t i o n o f t h e i n s e r t e d m o u s e D N A f r a g m e n t s . N u c l e o t i d e s e q u e n c i n g a n d D N a s e I f o o t p r i n t e x p e r i m e n t s s h o w e d t h a t T a n t i g e n b i n d s t o s i t e s in t h e s e f r a g m e n t s c o n s i s t i n g o f t w o t a n d e m l y o r i e n t e d G ( A ) A G G C p e n t a m e r s s e p a r a t e d b y A T r i c h s p a c e r s o f d i f f e r e n t l e n g t h s . T h e c e l l u l a r b i n d i n g s i t e s a r e v e r y s i m i l a r in t h e i r a r c h i t e c t u r e t o t h e S V 4 0 D N A b i n d i n g s i t e I. T h e i s o l a t e d c e l l u l a r D N A f r a g m e n t s w i t h T a n t i g e n b i n d i n g s i t e s o c c u r o n l y o n c e o r a f e w t i m e s in t h e m o u s e g e n o m e . O u r d a t a h e l p t o f u r t h e r d e f i n e t h e s t r u c t u r e o f T a n t i g e n ' s D N A b i n d i n g s i t e s . T h e g e n e t i c f u n c t i o n s o f t h e i s o l a t e d c e l l u l a r D N A e l e m e n t s a r e n o t k n o w n .
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Application of an immunoprecipitation procedure to the study of SV40 tumor antigen interaction with mouse genomic DNA sequences.
Simian Virus 40 (SV40) large T antigen is a DNA binding protein with high affinity for segments of the viral genome. To find out whether T antigen also binds to sequences of genomic cellular DNA we mixed T antigen and SAU 3 A restricted mouse DNA under stringent DNA binding conditions. Resulting protein-DNA complexes were immunoprecipitated using T antigen specific monoclonal or polyclonal anti...
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