Buitenwerf, Johannes

Welsh

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   University of GroningenRecombination in transformation of Bacillus subtilisBuitenwerf, JohannesIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionPublisher's PDF, also known as Version of recordPublication date:1977Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Buitenwerf, J. (1977). Recombination in transformation of Bacillus subtilis. s.n.CopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 08-06-2022SUMMARYT h i s  t h e s i s  d e s c r i b e s  e x p e r i m e n t s  a i m e d  a t  a d v a n c i n g  o u r  k n o w l e d g e  c o n -c e r n i n g  t h e  p r o c e s s  o f  t r a n s f o r m a t i o n  i n  B a c i L l u s  s u b t i L i s .  T o  t h i s  p u r -p o s e  m u t a n t s ,  i m p a i  r e d  i n  t r a n s f o r m a t i o n ,  w e r e  i s o l a t e d .  B o t h  i n  t h e s em u t a n t s  a n d  i n  t h e  h i g h l y - t r a n s f o r m a b l e  w i  l d - t y p e  s y n c h r o n o u s ' l y  t r a n s f o r -m i n g  a t  l o w  t e m p e r a t u r e t  t h e  f a t e  o f  t r a n s f o r m i n g  D N A  w a s  s t u d i e d .A f t e r  a  g e n e r a l  i n t r o d u c t i o n  i n  c h a p t e r  l ,  c o m p r i s i n g  a  b r i e f  r e -v i e w  o f  t h e  s a l i e n t  f e a t u r e s  o f  t h e  s y s t e m s  o f  t r a n s f o r m a t i o n  o f  B .  s u b -t i l i s  a n d  D ,  p n e w n o n i a e ,  i n  C h a p t e r  2  a  n u m b e r  o f  t r a n s f o r m a t i o n - d e f i c i e n tm u t a n t s  i s  d e s c r i b e d .  I n  t h e  w e l  l - t r a n s f o r m a b l e  w i  l d - t y p e ,  d o u b l e - s t r a n d e dd o n o r  D N A  i s  s u c c e s s i v e l y  c o n v e r t e d  i n t o  d o u b l e - s t r a n d e d  f r a g m e n t s ,  s i n g l e -s t r a n d e d  s e g m e n t s  a n d  i n t o  d o n o r - r e c i p i e n t  c o m p l e x .  M u t a n t  7 G - 7 3  c o n v e r t sd o u b l e - s t r a n d e d  f r a g m e n t s  i n t o  s i n g l e - s t r a n d e d  s e g m e n t s ,  b u t  o n l y  a  m i n o ra m o u n t  o f  d o n o r - r e c i p i e n t  c o m p l e x  i s  f o r m e d ,  T h e  d o n o r - r e c i p i e n t  c o m p l e xs h o w s  a n  a b e r r a n t  b u o y a n t  d e n s i t y  i n  C s C l - g r a d i e n t s  a s  c o m p a r e d  t o  d o n o r -r e c i p i e n t  c o m p l e x  f o r m e d  i n  t h e  w i  l d - t y p e .  M u t a n t  / G - 8 4  f o r m s  d o u b l e -s t r a n d e d  f r a g m e n t s  a n d  s i n g l e - s t r a n d e d  s e g m e n t s .  V i a  a n  i n t e r m e d i a t e  c o m -p l e x  o f  d o n o r  a n d  r e c i p i e n t  D N A  w i t h  a b e r r a n t  d e n s i t y ,  a  c o m p l e x  i s  f o r -m e d ,  w h i c h  i s  p h y s i c o - c h e m i c a l  l y i n d i s t i n g u i s h a b l e  f r o m  d o n o r - r e c i p i e n tc o m p l e x  f o r m e d  i n  t h e  w i  l d - t y p e ,  M u t a n t  / G - ! /  p r o d u c e s  d o u b l e - s t r a n d e df r a g m e n t s ,  b u t  t h e  d o n o r  D N A  i  s  n o t  p r o c e s s e d  f u r t h e r .B a s e d  o n  t h e  c o n j e c t u r e  t h a t  t h e  a b e r r a n t  b u o y a n t  d e n s i t y  d o n o r - r e -c i p i e n t  c o m p l e x  f o r m e d  i n  t h e  m u t a n t s  7 G - 7 3  a n d  7 G - 8 4  m i g h t  r e p r e s e n t  a ni n t e r m e d i a t e  i n  n o r m a l  r e c o m b i n a t i o n ,  t h e  c o n v e r s i o n  o f  w h i c h  i s  d e l a y e di n  t h e s e  m u t a n t s ,  C h a p t e r  3  d e a l s  w i t h  e x p e r i m e n t s  w h i c h  a r e  a i m e d  t oe x a m i n e  w h e t h e r  t h e  a b e r r a n t  b u o y a n t  d e n s i t y  d o n o r - r e c i p i e n t  c o m p l e x  i sa l s o  f o r m e d  i n  w i l d - t y p e  B ,  s u b t i L i s .  T o  t h i s  p u r p o s e  a  m e t h o d  w a s  u s e dt h a t  b o t h  s y n c h r o n i s e s  a n d  s l o w s  d o w n  t h e  t r a n s f o r m a t i o n  p r o c e s s .  S y n -c h r o n o u s  u p t a k e  o f  D N A  w a s  e f f e c t e d  b y  t h e  c h e l a t i n g  a g e n t  e t h y l e n e - d i a -m i n e - t e t r a - a c e t a t e  ( E D T A ) ,  w h i c h  p r e v e n t s  e n r r y ,  b u E  n o t  t h e  b i n d i n g  o ft r a n s f o r m i n g  D N A  t o  c o m p e t e n t  c e l l s .  A f t e r  t h e  a d d i t i o n  o f  e x c e s s  d i v a l e n tc a t i o n s  ( M g - ' )  s y n c h r o n o u s  e n t r y  a n d  c o n v e r s i o n  o f  t h e  b o u n d  D N A  i n t o  v a -r i o u s  m o l e c u l a r  f o r m s  i s  a c h i e v e d ,) aA d d i t i o n  o f  M g z *  i o n s  a t  1 7 o C  t o  c e l l s ,  l o a d e d  w i t h  d o n o r  D N A  i n  t h e  p r e -s e n c e  o f  E D T A  a t  3 0 o C ,  r e s u l t s  i n  t h e  f o r m a t i o n  o f  a  d o n o r - r e c i p i e n t  c o m -p l e x  t h a t  i s  e n d o w e d  b o t h  w i t h  a n  a b e r r a n t  b u o y a n t  d e n s i t y  a n d  l i t t l ' e8 1h i ^ l ^ ^ i . ^ l  ^ - + i . , i : . ,  A t + ^ -  - r i E r i ^ -  + L ^  + - ^ ^ ^ f ^ - - : ^ ^  ^ ^ l l -  r, u o ,  o e L , u , . y .  A f t e r  s h i f t i n g  t h e  t r a n s f o r r n i n g  c e ' l  l s  t o  3 0 " C ,  t h ea b e r r a n t  d o n o r - r e c i p i e n t  c o m p l e x  i s  c o n v e r t e d  i n t o  a  n o r m a l  b u o y a n t - d e n -s i  t y  c o m p l e x .C h a p t e r  4  d e a l s  w i t h  t h e  f u r t h e r  c h a r a c t e r i z a t i o n  o f  t h e  i n t e r m e d i a t er e c o m b i n a t i o n  c o m p l e x .  A l  k a l  i n e  s u c r o s e - g r a d  i e n t  c e n t r i  f u g a t  i  o n  s h o w e dt h a t  t h e  d o n o r  D N A  i n  t h e  i n t e r m e d i a t e  d o n o r - r e c i p i e n t  c o m o l e x  i s  c o v a -l e n t l y  b o n d e d  t o  t h e  r e c i p i e n t  D N A .  T r e a t m e n t  o f  t h e  i n t e r m e d i a t e  c o m p l e xw i t h  t h e  s i n g l e - s t r a n d e d  D N A  s p e c i f i c  n u c l e a s e  S 1  r e s u l t e d  i n  t h e  I  i b e r a -! i o n  o f  a c i d - s o i u b l e  p r o d u c t s .  l n  a d d i t i o n ,  t h e  b i o l o g i c a l  a c t i v i t y  o f  t h ec o m p l e x  i s  d e c r e a s e d  f o l l o w i n g  t r e a t m e n t  w i t h  n u c i e a s e  S 1 .  B o t h  o n  t h eb a s i s  o f  t h e s e  r e s u l t s  a n d  t h e  p r o p e r t i e s  o f  t h e  c o m p l e x  i n  C s C l - g r a -d i e n t s ,  i t  i s  a s s u m e d  t h a t  t h e  i n t e r m e d i a t e  d o n o r - r e c i p i e n t  c o n p l e x  i s  as t r u c t u r e  i n  w h i c h  a  s i n g l e - s c r a n d e d  s e g m e n t  o f  d o n o r  D N A  i s  p a r t i a l  l yp a i r e d  w i t h  t h e  r e c i p i e n t  D N A  a n d  i s  b o u n d  c o v a l e n t i y  t o  t h e  r e c i p i e n tD N A  a t  t h e  p a i  r e d  e x t r e m i t y .82

Recombination in transformation of Bacillus subtilis

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Recombination in transformation of Bacillus subtilis

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