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Isolation and characterization of restriction endonucleases from Fusobacterium nucleatum Lui, Ann Chin Par 1978

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ISOLATION AND CHARACTERIZATION OF RESTRICTION ENDONUCLEASES FROM Fusobaotevium nucleatum by ANNE CHIN PAR/LUI B.Sc. , U n i v e r s i t y of B r i t i s h C o l u m b i a , 1976 A:THESIS SUBMITTED IN:PARTIAL : FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES Department of B i o c h e m i s t r y The U n i v e r s i t y of B r i t i s h Co lumbia We a c c e p t t h i s t h e s i s as c o n f o r m i n g to the r e q u i r e d s t a n d a r d THE UNIVERSITY OF BRITISH COLUMBIA Augu s t , 1978 (c) Anne C .P . J Lu i In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Head o f my Department or by h i s r e p r e s e n t a t i v e s . I t i s unde r s tood t h a t c o p y i n g or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department of B i o c h e m i s t r y The U n i v e r s i t y of B r i t i s h Co lumbia 2075 Wesbrook P l a c e Vancouve r , Canada V6T 1W5 Augu s t , 1978. - -ii -ABSTRACTS F i v e s t r a i n s of Fusobaotevium nuol.eatum were s c reened f o r the p re sence o f r e s t r i c t i o n enzymes and the f o l l o w i n g r e c o g n i t i o n s p e c i f i c i t i e s and c l e a v a g e s i t e s ( i n d i c a t e d by the ar rows \ ) were c h a r a c t e r i z e d : Fnu A l , 5 ' -G -ANTC -3 1 ; Fnu C J , 5 ' - ^GATC-S ' ( u n m o d i f i e d bases o n l y ) ; Fnu DI, S ' - G G ^ C C - S ' ; Fnu D11 , 5 1 -CG + CG-3 ' ; Fnu D i l i , 5 ' - G C G ^ C - 3 ' ; Fnu E I , 5 ' - GATC-3 1 (both m o d i f i e d and u n m o d i f i e d bases r e c o g n i z e d ) ; Fnu 481, 5 1 - T C ^ G A - 3 1 . - H i -TABLE OF CONTENTS Page ABSTRACT .ii TABLE OF CONTENTS Hi LIST OF TABLES vi LIST OF FIGURES vii ACKNOWLEDGEMENTS ix ABBREVIATIONS x INTRODUCTION 1 Nomenc la ture 8 METHODS AND MATERIALS 9 The b a c t e r i a 9 1. I s o l a t i o n o f Fusobacterium nucleatum 9 2. C h a r a c t e r i z a t i o n o f Fusobacterium -nuoleatum 9 3. Growth c o n d t i i o n s of the b a c t e r i a 10 P u r i f i c a t i o n o f r e s t r i c t i o n enzymes 10 1 . Crude e x t r a c t s 10 2. B i o g e l column 11 3. P h o s p h o c e l 1 u l o s e column 11 4. D E A E - c e l 1 u l o s e column 11 5. C o n c e n t r a t i o n 12 6. Assay 12 - iv -Page C h a r a c t e r i z a t i o n of r e s t r i c t i o n enzymes 13 1. D i g e s t i o n of doub le s t r a n d e d b a c t e r i o p h a g e DNA . . . . 1 3 2. 2-D e l e c t r o p h o r e s i s and homochromatography 14 a) [ y S 2P]-ATP exchange 15 b) Purification of labelled fragments 16 c) Digestion with PI enzyme 16 d) Electrophoresis on cellulose acetate .17 e) Homochromatography 17 f) Determination of 5'-terminus 18 3. D e t e r m i n a t i o n of r e s t r i c t i o n enzyme c l e a v a g e s i t e s 1 9 4. C leavage mapping by n e a r e s t ne i ghbou r a n a l y s i s . . . . 20 a) Preparation of restriction fragments 20 b) Mapping of DNA fragments by "nearest neighbour" ": analysis 21 P r e p a r a t i o n of <i>X174am3 DNA 22 T.. Phage growth 22 2. P u r i f i c a t i o n o f RF DNA 23 3. P u r i f i c a t i o n of v i r a l ( + ) s t r a n d DNA 24 RESULTS 26 The b a c t e r i a 26 Fnu D 26 1 . P u r i f i c a t i o n 26 2. C h a r a c t e r i z a t i o n of Fnu DI, Fnu D I I , Fnu D i l i 32 . . a) Effect of salt concentration 32 b) Stability 32 c)Double digestion analy sis of Fnu DI and Fnu Dili. 3 2 - V -Page d) Sequencing cleavage sites of Fnu DI and Fnu Dili 32 e) Double digestion analysis of Fnu DII 36 f) Cleavage mapping of Fnu DII by nearest neighbour analysis 36 g) Sequencing cleavage sites of Fnu DII 40 Fnu EI 42 1. P u r i f i c a t i o n 42 2. C h a r a c t e r i z a t i o n 42 a) Double digestion analysis 42 b) Sequencing the recognition site by 2-D fraction-ation 43 a) Digestion of fl RF DNA 52 Fnu CI 54 Fnu A l and Fnu A l l 54 Fnu 48 57 M i s c e l l a n e o u s 64 DISCUSSION 66 REFERENCES 76 - vi -LIST OF TABLES Page Tab le 1. R e s u l t s of n e a r e s t ne i ghbou r a n a l y s i s compared w i t h computer p r d i c t i o n s 39 Tab le 2. Double d i g e s t i o n o f SI 3 RF DNA w i t h Fnu EI and one o f the f o l l o w i n g enzymes 48 Tab le 3. D e t e r m i n a t i o n of the 5 ' - t e r m i n a l base f o l l o w i n g c l e a v a g e by Fnu EI 50 Tab l e 4. Double d i g e s t i o n o f <£>X RF DNA w i t h Fnu 48 and one o f the f o l l o w i n g enzymes 62 Tab le 5. The s i z e s : o f f ragments gene ra ted from c l e a v a g e of $X DNA by Fnu enzymes 68 - vii -LIST OF FIGURES Page F i g . 1. Diagram showing the s e t - u p f o r t r a n s f e r r i n g m a t e r i a l f rom ee l 1u l o se a c e t a t e s t r i p to DEAE-cel 1 u l ose shee t ,1:8 F i g . 2. As say o f f r a c t i o n s f rom B i o g e l A 0.5m column o f Fnu D r e s t r i c t i o n enzymes a n a l y z e d by ge l e l e c t r o p h o r e s i s 29 F i g . 3. Assay o f f r a c t i o n s f rom p h o s p h o c e l l u l o s e column of Fnu D r e s t r i c t i o n enzymes w i t h ge l e l e c t r o -p h o r e s i s 30 F i g . 4. '-Assay of f r a c t i o n s f rom DEAE-Sephadex A-50 column of Fnu DI and Fnu D11 w i t h gel e l e c t r o p h o r e s i s 31 F i g . 5. A u t o r a d i o g r a p h o f the ge l f o r the d e t e r m i n a t i o n o f the c l e a v a g e s i t e of Fnu DI 35 F i g . 6. A c r y l a m i d e gel p a t t e r n s o f 4>X RF DNA d i g e s t e d w i t h the f o l l o w i n g s p e c i f i c endonuc l ea se s . . . . 37 F i g . 7. A u t o r a d i o g r a p h of n e a r e s t ne i ghbou r a n a l y s i s f o r d e t e r m i n i n g the c l e a v a g e map o f Fnu DI I on *X DNA 38 F i g . 8. A u t o r a d i ograph o f Fnu DII c l e a vage s i t e charact<- . e r i z a t i o n by sequence a n a l y s i s 41 F i g . 9. Gel e l e c t r o p h o r e s i s a n a l y s i s o f a s say s o f f r a c t i o n s f rom p h o s p h o c e l 1 u l o s e column of Fnu EI 44 F i g . 10. Gel e l e c t r o p h o r e s i s a n a l y s i s o f a s say s o f > f r a c t i o n s f rom D E A E - c e l 1 u l o s e column of Fnu EI 45 F i g . 11. Compar i son of Fnu EI w i t h Mbo I d i g e s t i o n p a t t e r n s of v a r i o u s DNAs on 5% a c r y l a m i d e g e l s 46 F i g . 12. Double d i g e s t i o n o f S13 RF DNA w i t h Fnu EI and the v a r i o u s enzymes 47 F i g . 13. C leavage map of Fnu EI on S13 DNA 49 - viii -Page F i g . 14. A u t o r a d i o g r a p h of the 2-D f r a c t i o n a t i o n of o l i g o n u c l e o t i d e s o b t a i n e d from Pi p a r t i a l d i g e s t i o n o f X DNA r e s t r i c t i o n f ragments p roduced by Fnu EI 51 F i g . 15. D i g e s t i o n of f l RF DNA 53 F i g . 16. A u t o r a d i o g r a p h of Fnu A l c l e a v a g e c h a r a c t e r -i z a t i o n by sequence a n a l y s i s 56 F i g . 17. Gel e l e c t r o p h o r e s i s of a s say s of f r a c t i o n s from B i o g e l column of Fnu:.48 58 F i g . 18. Assays of f r a c t i o n s from p h o s p h o c e l 1 u l o s e column of Fnu 48 a n a l y z e d by gel e l e c t r o p h o -r e s i s 59 F i g . 19. Assay o f f r a c t i o n s f rom DEAE-Sephadex column o f Fnu 48 by ge l e l e c t r o p h o r e s i s a n a l y s i s . . 60 F i g . 20. Double d i g e s t i o n o f RF DNA w i t h Fnu 48 and v a r i o u s enzymes 61 F i g . 21. A u t o r a d i o g r a p h of the gel f o r d e t e r m i n a t i o n of c l e a v a g e s i t e o f Fnu 48 by Brown and Smi th method 63 F i g . 22. Assay o f f r a c t i o n s f rom B i o g e l column o f the h igh speed s u p e r n a t a n t of Bacteroides melano-genicus c e l l e x t r a c t s 65 F i g . 23. C leavage map o f Fnu enzymes on <f>X174am3 DNA 70 - ix -ACKNOWLEDGEMENTS I w i s h to thank C a r o l f o r i n v a l u a b l e c r i t i c i s m s on the t h e s i s ; Rosa f o r spend ing many l a t e n i g h t s t y p i n g ; Mary, H e a t h e r , Debb i e , H e l e n , Bob M i l l e r , S h i r l e y and C a r o l f o r e x p e r t t e c h n i c a l a d v i c e ; D a v i d , Bob,and Anne f o r h e l p f u l d i s c u s s i o n ; and most of a l l , Dr. B a r r y C. McBr ide and Dr. M i c h a e l Smith f o r t h e i r e x c e l l e n t s u p e r v i s i o n . I am e s p e c i a l l y g r a t e f u l to Dr. M. Smith f o r h i s d i r e c t g u i d a n c e , encouragement , c o n s i d e r a t i o n and u n d e r s t a n d i n g . The M e d i c a l Research C o u n c i l i s thanked f o r g r a n t i n g me a S t u d e n t s h i p i n the p e r i o d 1977-1978. - X -ABBREVIATIONS A amps, mamps ATP dATP, dCTP, dGTP, dTTP C C i cpm DEAE DNA EDTA G x g M, mM, yM N rpm T T r i s V deoxyadenos i ne amperes, mi 1 1 i ampe re s 5 ' - t r i p h o s p h a t e r i b o n u c l e o s i d e of aden ine 5 ' - t r i p h o s p h a t e d e o x y r i b o n u c l e o s i d e s of a d e n i n e , c y t o s i n e , guan ine and thymi ne d e o x y c y t i d i n e C u r i e count s per minute d i e t h y l ami n o e t h y l d e o x y r i b o n u c l e i c a c i d 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 deoxyguanos ine t imes the f o r c e of g r a v i t y mo lar (moles per l i t e r ) , m i l l i m o l a r , mi c r o m o l a r any o f the f o u r d e o x y r i b o n u c l e o s i d e s r e v o l u t i o n s per minute deoxythymi d i ne t r i s ( h y d r o x y m e t h y l ) a m i nome thane vo l t s - 1 -INTRODUCTION The term " r e s t r i c t i o n " comes f rom g e n e t i c s t u d i e s o f i n t e r a c t i o n between b a c t e r i o p h a g e s and t h e i r h o s t s . The f i r s t such s tudy was r e p o r t e d by L u r i a and Human ( 1952 ) . They found t h a t phages l i b e r a t e d from some b a c t e r i a l mutants a c q u i r e new hos t range c h a r a c t e r i s t i c s wh ich are l o s t upon r e - i n f e c t i o n of p e r m i s s i v e ho s t s t h a t a re g e n o t y p i c a l l y d i f f e r e n t f rom the mu tan t s . L a t e r , A r be r and Dus so i x (1962) proposed a DNA m o d i f i c a t i o n and r e s t r i c t i o n mechanism i n E. coli B and K to e x p l a i n these f i n d i n g s . They sugges ted t h a t r e s t r i c t i o n o c c u r s when i n c o m p a t i b l e DNA e n t e r i n g the b a c t e r i u m i s r e c o g n i z e d and degraded to v a r i o u s f r agment s whereas hos t DNA i s p r o t e c t e d from t h i s d i g e s t i o n by m o d i f i c a t i o n o f the r e c o g n i t i o n s i t e (Du s so i x & A r b e r , 1962 ) . G e n e t i c e x p e r i m e n t s p r o v i d e d the f i r s t e v i d e n c e o f s i t e . s p e c i f i c r e c o g n i t i o n i n m o d i f i c a t i o n and r e s t r i c t i o n of DNA ( f o r r e v i e w , see A r b e r & L i n n , 1969) . Sequence a n a l y s i s u s i n g the method o f Weiss and R i c h a r d s o n (1967) has shown t h a t r e c o g n i t i o n i n v o l v e s s p e c i f i c n u c l e o l y t i c sequences ( K e l l y & S m i t h , 1970 ) . R e s t r i c t i o n enzymes can t h e r e f o r e be d e f i n e d as s i t e s p e c i f i c d e o x y r i b o e n d o n u c l e a s e s t h a t r e c o g n i z e s p e c i f i c n u c l e o t i d e sequences i n DNA mo iecu !e s .and produce d o u b l e -s t r a n d e d c l e a v a g e s to g i v e a f i n i t e number of f r a g m e n t s . - 2 -There are two c l a s s e s of r e s t r i c t i o n e n d o n u c l e a s e s . The E. coli B and K enzymes be long to the C l a s s I r e s t r i c t i o n endonuc lea se s wh ich r e c o g n i z e s p e c i f i c s i t e s on the DNA but c l e a v e i t at random p o i n t s . C o f a c t o r s , ATP, M g + + and S-a d e n o s y l m e t h i o n i n e are r e q u i r e d . C l a s s II r e s t r i c t i o n enzymes a l s o r e c o g n i z e s p e c i f i c s i t e s on the DNA but they c l e a v e the DNA s p e c i f i c a l l y to g i v e d i s c r e t e f ragment s s e p a r a b l e by ge l e l e c t r o p h o r e s i s and most of them r e q u i r e o n l y M g + + f o r a c t i v i t y . The d i s c o v e r y of these C l a s s II endonuc l ea se s has r e v o l u t i o n a r i z e d the methods f o r a n a l y z i n g DNA s t r u c t u r e and f u n c t i o n . Subsequent to the p u r i f i c a t i o n of one such enzyme from Haemophilus influenzae s e r o t y p e d ( Smi th & W i l c o x , 1970) and i t s a p p l i c a t i o n i n o b t a i n i n g SV40 DNA f r agment s (Danna & Nathans , 1971 ) , the a v a i l a b i l i t y o f s p e c i f i c f r agment s has e x p e d i t e d DNA a n a l y s i s i n g e n e r a l . Danna et al (1973) were the f i r s t to o b t a i n a r e s t r i c t i o n c l e a v a g e map o f the d o u b l e -s t r a n d e d SV40 genome. C leavage l o c a t i o n s p r o v i d e d p h y s i c a l r e f e r e n c e s and se rve as a framework f o r mapping s t u d i e s . Thus c l e a v a g e map d e t e r m i n a t i o n has e v o l v e d to be an i n d i s -p e n s i b l e p r ocedu re i n genome a n a l y s i s and an i n c r e a s i n g volume of the work i s done on a wide range o f DNAs, f rom b a c t e r i o - . phage DNA to human m i t o c h o n d r i o n DNA ( f o r r e v i e w , see R o b e r t s , 1976 ) . E s t a b l i s h i n g marker s i t e s i s o n l y one o f the many uses - 3 -o f r e s t r i c t i o n enzymes. T r a n s c r i p t i o n a l mapping i s a l s o amenable to a n a l y s i s u s i n g s i t e s p e c i f i c endonuc l ea se s i n e xpe r imen t s such as marker r e s cue and e x c i s i o n a l d e l e t i o n s ( L a i & Nathans , 1974) . F u r t h e r m o r e , r e s t r i c t i o n endonuc l ea se s a re i n s t r u m e n t a l i n the deve lopment of the p r e s e n t r a p i d methods of DNA sequence a n a l y s i s (Sanger et al, 1977a, 1977b; Maxam & G i 1 b e r t , 1 977) , a l l o w i n g g r e a t p r o g r e s s i n the s t udy of g e n e t i c c o n t r o l mechanisms and gene o r g a n i z a t i o n . R e l a t i v e l y more obscu re uses o f t he se enzymes i n c l u d e d e t e r m i n a t i o n o f p h y l o g e n i c r e l a t i o n s h i p s ( P o t t e r et al, 1975) and the d e t e c t i o n of c e l l c u l t u r e c o n t a m i n a t i o n (Grossman et air, 1 977 ) . One of the most c o n t r o v e r s i a l a p p l i c a t i o n s of r e s t r i c t i o n enzymes i n v o l v e s the in vitro c o n s t r u c t i o n o f c h i m e r i c DNA i n t r o d u c e d by Cohen et al ( 1973 ) . The s i t e s p e c i f i c endonuc l ea se s have t h e r e f o r e p i o n e e r e d the f i e l d o f g e n e t i c e n g i n e e r i n g which p romi ses major advances i n both h e a l t h and b a s i c s c i e n c e s ( f o r a r e v i ew o f endonuc lea se s and r ecomb inan t m o l e c u l e s see R o b e r t s , 1977 ) . The v e r s a t i l i t y o f these endonuc l ea se s i n the advancement o f DNA s t r u c t u r e a n a l y s i s has r e s u l t e d i n an i n s a t i a b l e demand f o r enzymes of d i f f e r i n g s p e c i f i c i t i e s . I t i s t h e r e f o r e no s u r p r i s e t h a t g r e a t a t t e n t i o n i s f o c u s e d . o n the s ea r ch f o r more r e s t r i c t i o n enzymes. At p r e s e n t , r e s t r i c t i o n a c t i v i t i e s are found i n over 150 s t r a i n s of a e r o b i c and f a c u l t a t i v e b a c t e r i a w i t h 42 d i f f e r e n t r e c o g n i t i o n sequences i d e n t i f i e d . S i n c e many enzymes i s o l a t e d - 4 -from d i f f e r e n t s ou r ce s e x h i b i t the same r e c o g n i t i o n s p e c i f i c i t y the term " i s o s c h i z o m e r " has been i n t r o d u c e d ( R o b e r t s , 1976) f o r such enzymes. The Bacillus and Haemophilus genera a re the most p r o d u c t i v e w i t h 37 and 28 enzymes i s o l a t e d r e s p e c t i v e l y . B a c t e r i a a re not the o n l y sou rce s of s i t e s p e c i f i c n u c l e a s e s . R e s t r i c t i o n p r o p e r t i e s are known to be p r e s e n t i n the b a c t e r i o p h a g e P i ( A rbe r & D u s s o i x , 1962) and the enzyme r e s p o n s i b l e has been p u r i f i e d (Haberman, 1974) . S i t e s p e c i f i c a c t i v i t i e s a re a l s o found i n the e u k a r y o t i c green a l g a e , Chlamydomonas ( Bu r ton et al, 1977) and i n A f r i c a n green monkey c e l l s (Brown et al, 1978 ) . However t he se enzymes f rom e u k a r y -o te s produce s i n g l e - s t r a n d e d gaps i n s t e a d o f d o u b l e - s t r a n d e d b r e a k s , thus l e a v i n g the p r o k a r y o t e s as the major s ou rce f o r C l a s s II r e s t r i c t i o n enzymes. The m a j o r i t y of the C l a s s II r e s t r i c t i o n enzymes r e c o g -n i z e p a l i n d r o m i c o l i g o n u c l e o t i d e s f o u r to s i x bases l ong i n d o u b l e - s t r a n d e d DNA. An e x c e p t i o n to the t w o f o l d sequence symmetry i s found i n a f a m i l y o f enzymes s i m i l a r to the a c t i v i t y i s o l a t e d from Haemophilus gallinarium t h a t r e c o g n i z e s a symmetr i c p e n t a n u c l e o t i d e s . Duplex s t r u c t u r e i s n e c e s s a r y f o r . r e c o g n i t i o n by most r e s t r i c t i o n endonuc l ea se s . However c l e a v a g e s i n s i n g l e - s t r a n d e d DNA a re found i n s e v e r a l i n s t a n c e s s u g g e s t i n g d i r e s t r e c o g n i t i o n o f s i n g l e - s t r a n d e d sequences ( H o r i u c h i & Z i n d e r , 1975 ) . Other a u t h o r s a t t r i b u t e the appa ren t s i n g l e - s t r a n d e d c l e a v a g e to the p re sence o f dup l e x - 5 -r e g i o n s g ene r a t ed by secondary s t r u c t u r e s a t the r e c o g n i t i o n s i t e ( B l a k e s l e y & W e l l s , 1975 ) . In a d d i t i o n to the r e c o g -n i t i o n sequence, n e i g h b o u r i n g n u c l e o t i d e s may a f f e c t the k i n e t i c s o f c l e a vage (Thomas & D a v i s , 1975 ) . S c h e l l e r et al (1977) ob se r ved t h a t r e c o g n i t i o n s i t e s o c c u r i n g a t the ends o f the m o l e c u l e a re not c l e a v e d and t h a t most enzymes are c a p a b l e o f r e c o g n i z i n g s y n t h e t i c d e c a n u c l e o t i d e s t h a t c o n -t a i n r e s t r i c t i o n s i t e s near the c e n t r e of the m o l e c u l e s . T h i s sugges t s t h a t t o p o l o g i c a l c o n s t r a i n t s w i t h i n the DNA m o l e c u l e are not r e q u i r e d i n t he se i n s t a n c e s . The e x a c t r e c o g n i t i o n mechanisms o f s p e c i f i c endonuc l ea se s remain o b s c u r e . S e v e r a l c l e a v a g e p r o p e r t i e s have s u r f a c e d from the work on the c h a r a c t e r i z a t i o n of r e s t r i c t i o n enzymes wh ich make d o u b l e - s t r a n d e d s c i s s i o n s to y i e l d f r agment s of DNA w i t h 5 ' - p h o s p h o r y l and 3 ' - h y d r o x y l t e r m i n i . The c l e a v a g e may be n o n - s p e c i f i c as i n the case w i t h C l a s s I r e s t r i c t i o n enzymes o r , as w i t h C l a s s I I enzymes, the c l e a v a g e may be a t a s i t e w i t h i n , o r a t d e f i n e d d i s t a n c e s f r o m , the r e c o g n i t i o n sequence. An example o f c l e a v a g e o u t s i d e o f the r e c o g n i t i o n sequence i s found i n the enzyme from Haemophilus gallinarium where c l e a v a g e o c c u r s a t f i v e n u c l e o t i d e s 5' from the sequence 5 1 - .GACGC-3 ' . In the case o f d e f i n e d c l e a v a g e s , the r e s u l t a n t f r agment s are e i t h e r f l u s h - e n d e d or have c o h e s i v e ends w i t h - 6 -3' or 5 ' e x t e n s i o n s depend ing on the enzyme used . A r e c e n t r e v i ew by Robe r t s (1976) p r o v i d e s a thorough d i s c u s s i o n on a l l a s p e c t s o f r e s t r i c t i o n enzymes. A l t h o u g h a e r o b i c b a c t e r i a have been e x t e n s i v e l y s u r veyed f o r s i t e s p e c i f i c e n d o n u c l e a s e s , l i t t l e a t t e n t i o n had been g i ven to the anae robe s . We have examined the o b l i g a t e anae robe , Fusobaoterium, i s o l a t e d from the human o r a l c a v i t y , and have found r e s t r i c t i o n enzymes to be u b i q u i t o u s i n t h i s genus. Fusobaoterium a re g r a m - n e g a t i v e n o n - s p o r u l a t i n g rods b e l o n g i n g to the f a m i l y Baoteroidacenae ( B e r g e y ' s Manua l , 8 th ed. ) A chemoorganot roph , Fusobaoterium p roduces b u t y r i c a c i d as the major p r o d u c t from pep tone , a l t h o u g h v a r i o u s s p e c i e s produce i n d o l e as w e l l . In s c r e e n i n g f o r r e s t r i c t i o n enzymes i n a n a e r o b i c b a c t e r i a , we have chosen the p r ocedu re o f Smith & W i l c o x (1970) i n t h e i r p u r i f i c a t i o n of the enzyme from Haemophilus influenzae. Enzymes t h a t a re used as t o o l s f o r s t u d y i n g DNA need o n l y be f r e e f rom n o n - s p e c i f i c n u c l e a s e s . I t f o l l o w s t h a t the p u r i f i c a t i o n of r e s t r i c t i o n endonuc l ea se s i n v o l v e s removal o f n u c l e i c a c i d s , c o n t a m i n a t i n g n u c l e a s e s and s e p a r a t i o n o f d i f f e r e n t r e s t r i c t i o n a c t i v i t i e s . N u c l e i c a c i d s can be removed by B i o g e l ch romatog raphy , a d s o r p t i o n to h y d r o x y a p a t i t e (Kopecka , 1975) , s t r e p t o m y c i n s u l f a t e p r e c i p i t a t i o n or p o l y e t h y l e n e i m i n e p r e c i p i t a t i o n ( B i c k l e et al, 1977) . We have used the B i o g e l column f i r s t to remove n u c l e i c a c i d s and to e f f e c t some s i z e s e p a r a t i o n , - 7 -thus a l l o w i n g the d e t e c t i o n o f r e s t r i c t i o n a c t i v i t y i n the c rude e x t r a c t s a f t e r a s i n g l e c h r o m a t o g r a p h i c s t e p . F u r t h e r p u r i f i c a t i o n i n v o l v e s i on - exchange ch romatog raphy . A f f i n i t y chromatography on h e p a r i n - a g a r o s e column i s o f t e n e f f e c t i v e i n removing c o n t a m i n a t i n g n o n - s p e c i f i c n u c l e a s e s ( B i c k l e et al, 1977 ) . F i v e s t r a i n s of Fuse-bacterium nucleatum have been s c reened f o r the p re sence o f r e s t r i c t i o n enzymes i n the B i o g e l column e l u e n t s . E n d o n u c l e o l y t i c a c t i v i t i e s have been found i n a l l the s t r a i n s examined. The p u r i f i c a t i o n and c h a r a c t e r i z a t i o n o f these enzymes w i l l be d e s c r i b e d . - 8 -Nomenc lature The system o f nomenc la tu re most commonly used f o r r e s t r i c t i o n endonuc l ea se s i s g i v en by Smith and Nathans ( 1973 ) . Th i s system u t i l i z e s a t h r e e - l e t t e r i t a l i c i z e d a b b r e v i a t i o n d e r i v e d from the f i r s t l e t t e r of the genus and the f i r s t two l e t t e r s o f the s p e c i e s f rom which the enzymes i s i s o l a t e d . For example , the enzyme f rom Haemophilus influenzae i s c a l l e d Hin. S t r a i n or e x t r a -chromosomal i d e n t i f i c a t i o n i s denoted by a n o n - i t a l i c i z e d f o u r t h l e t t e r e . g . H. influenzae s e r o t y p e d becomes Hind; E. ooli l y s o g e n i c f o r PI becomes Eco?. Roman numera l s a re used when more than one enzyme i s found i n the same o r gan i sm. Thus t h e r e are Hind I, Hind II and Hind I I I . S i m i l a r l y , enzymes f rom Fusobaoterium nucleatum s t r a i n D are c a l l e d FnuD I, FnuD I I and FnuD I I I . The DNA f ragment s produced by r e s t r i c t i o n enzymes are a s s i g n e d A r a b i c numera l s i n o r d e r of i n c r e a s i n g m o b i l i t y on ge l e l e c t r o p h o r e s i s . - 9 -METHODS & MATERIALS The b a c t e r i a A l l the b a c t e r i a l s t r a i n s used i n t h i s t h e s i s were i s o l a t e d and c h a r a c t e r i z e d i n the l a b o r a t o r y o f Dr. B.C. McBr ide (Department o f M i c r o b i o l o g y , U n i v e r s i t y of B r i t i s h Columbi a ) . 1. I s o l a t i o n o f Fusobaoterium tiucleatum . Samples f rom g i n g i v a l c r e v i c e s of the human o r a l c a v i t y were s t r e a k e d onto b l ood (human) agar p l a t e s c o n t a i n -i n g 0.001% (.w/v) c r y s t a l v i o l e t and i n c u b a t e d a t 37°C i n an a n a e r o b i c chamber (Coy M a n u f a c t u r i n g ) f i l l e d w i t h 85% n i t r o g e n , 5% CO2 and 10% hydrogen. C o l o n i e s w i t h r od - shaped b a c t e r i a t h a t g r e w . w e l l under such c o n d i t i o n s were i n n o c u l a t e d i n t o media c o n t a i n i n g 1.7% (w/v) t r y p t i c a s e (BBL. B e c t o n , D i c k i n s o n & C o . ) , 0.3% (W/v) y e a s t e x t r a c t ( D i f c o ) , 0.5% (w/v) NaCI, 0.25% (w/v) K^HPO^ 0.25% (w/v) g l u c o s e , 5 ug/ml hemin, pH 7 and a l l o w e d to grow a t 37°C i n the a n a e r o b i c chamber. 2. C h a r a c t e r i z a t i o n of Fusobaoterium nucleatum The c u l t u r e s were t e s t e d f o r v i a b i l i t y on agar p l a t e s i n c u b a t e d a e r o b i c a l l y and f o r the p r o d u c t i o n of b u t y r i c a c i d , i n d o l e and c a t a l a s e . C u l t u r e s t h a t were c h a r a c t e r i z e d were s t o r e d f r o z e n a t -70°C i n the e x p o n e n t i a l phase i n the p re sence of 7% DMSQ i n one ml vo lumes. Under t he se c o n d i t i o n s , the b a c t e r i a - 10 -remained v i a b l e f o r up to 1-2 y e a r s . 3. Growth c o n d i t i o n s o f the b a c t e r i a Frozen c u l t u r e s of Fu sobacte r iu rn were i n n o c u l a t e d i n t o 10 ml o f med ia , i n c u b a t e d a t 37°C i n the a n a e r o b i c chamber to s t a t i o n a r y growht phase ( app rox . 48h ) , t r a n s f e r r e d to 200 ml o f media and grown i n a s i m i l a r manner. The 200 ml c u l t u r e s were used to i n n o c u l a t e 1.8 I o f med ia . The f i n a l 2 I c u l t u r e s were grown out of the chamber a t 37°C i n t i g h t l y s t oppe red 2 I e r l enmeye r f l a s k s . The head space between the rubber s topped and the f l u i d l e v e l s hou l d be kept to a minimum i n o r d e r to o b t a i n r e l a t i v e l y a n a e r o b i c c o n d i t i o n s . C e l l s f rom a c u l t u r e i n the s t a t i o n a r y growth phase ( i n c u b a t e d f o r a p p r o x i m a t e l y 48h w i t h a 10% v/v i nnocu lum) were h a r v e s t e d and kept f r o z e n at - 20°C . Between 2-3 g of c e l l s per l i t r e o f media were o b t a i n e d . P u r i f i c a t i o n o f r e s t r i c t i o n enzymes T. Crude e x t r a c t s Between 6 to 10 g of f r o z e n c e l l s were used f o r the i s o l a t i o n o f r e s t r i c t i o n enzymes. A l l p u r i f i c a t i o n p r o c e d u r e s were c a r r i e d out a t 4°C. The c e l l s were re suspended i n 12 to 20 ml o f b u f f e r A (0.01 M T r i s - H C l , pH 7 .5 , 0.01 M 2-mer-c a p t o e t h a n o l ) and s o n i c a t e d (about 10 x 20 sec ) u n t i l most o f t he c e l l s were broken as d e t e r m i n e d by e x a m i n a t i o n f o r the absence o f f i l a m e n t o u s s t r u c t u r e s i n a phase c o n t r a s t m i c r o -- 1J: -s cope . The s o n i c a t e d p r e p a r a t i o n was c e n t r i f u g e d a t 100,000 x g f o r 90 min i n a Beckman type 35 r o t o r to remove c e l l u l a r d e b r i s . 2. B i o g e l column The s u p e r n a t a n t f rom the h igh speed p e n t r i f l i g a t i o n was a d j u s t e d to 1 M NaCI and a p p l i e d to a 25 x 500 mm B i o g e l A 0.5 m ( B i o R a d , 100-200 mesh) column p r e - e q u i 1 i b r a r e d w i t h b u f f e r B (1 .0 M NaCI, 0.1 M T r i s - H C l , pH 7 .5 , 0.01 M 2-mer-c a p t o e t h a n o l ) . The sample was e l u t e d from the column w i t h a b u f f e r B a t a f l o w r a t e of a p p r o x i m a t e l y 1 m l/min . S i x t y 5 ml f r a c t i o n s were c o l l e c t e d and 5 u l a l i q u o t s o f s e l e c t e d f r a c t i o n s as sayed f o r s i t e s p e c i f i c e n d o n u c l e o l y t i c a c t i v i t y (see s e c t i o n on a s s a y s ) . A l l f r a c t i o n s c o n t a i n i n g p o t e n t i a l s i t e s p e c i f i c endonuc lea se a c t i v i t y were poo l ed and d i a l y z e d a g a i n s t b u f f e r C (10% v/v g l y c e r o l , 0.01 M KHP0 4 , pH 7.4, 0.01 M 2 - m e r c a p t o e t h a n o l , 1 0 " 4 M EDTA). 3. P h o s p h o c e l 1 u l o s e column The d i a l y z e d f r a c t i o n s were a p p l i e d to a 25 x 150 mm p h o s p h o c e l l u l o s e ( S c h l e i c h e r & S c h u e l l ) c o l u m n p r e - e q u i 1 i b r a t e d w i t h b u f f e r C and e l u t e d w i t h 200 ml of b u f f e r C c o n t a i n i n g a l i n e a r g r a d i e n t o f KC1 (0 to 1.0 M) a t a f l o w r a t e of a p p r o x i -ma te l y 1 m l/min . F i v e ml f r a c t i o n s were c o l l e c t e d and 5 y l a l i q u o t s a s s a y e d . 4. DEAE c e l l u l o s e column A c t i v e f r a c t i o n s f rom the p r e v i o u s column were combined - 12 -and d i a l y z e d a g a i n s t b u f f e r C and a p p l i e d to a 12 x 100 mm DEAE c e l l u l o s e (DE 22, Whatman) column p r e - e q u i 1 i b r a t e d w i t h b u f f e r C and e l u t e d w i t h 200 ml o f b u f f e r C c o n t a i n i n g a l i n e a r g r a d i e n t of KC1 a t a f l o w r a t e of a p p r o x i m a t e l y 0.5 m l /m in . The e x a c t g r a d i e n t used v a r i e d w i t h d i f f e r e n t enzyme p r e p a r a -t i o n s . F i v e ml f r a c t i o n s were c o l l e c t e d and 5 y l a l i q u o t s a s s a yed . The DEAE c e l l u l o s e i n t h i s ch romatography s tep c o u l d be s u b s t i t u t e d by s i m i l a r column m a t e r i a l s w i t h more r e s o l v i n g power such as the DEAE Sephadex (A -50 , P h a r m a c i a , 40-120y) and DEAE-Sephacel ( P h a r m a c i a ) . 5. C o n c e n t r a t i o n The p u r i f i e d f r a c t i o n s were c o n c e n t r a t e d e i t h e r by d i a l y s i s i n b u f f e r C c o n t a i n i n g 50% g l y c e r o l (v/v) o r by a d s o r p t i o n to i on -exchange co lumns . C o n c e n t r a t i o n u s i n g the chromatography method i n v o l v e s e l u t i o n from a 12 x 60 mm column c o n t a i n i n g e i t h e r D E A E - c e l 1 u l o s e (DE 22, Whatman) o r p h o s p h o c e l 1 u l o s e by b u f f e r C i n 1 M KC1. G l y c e r o l was then added to the f r a c t i o n s w i t h r e s t r i c t i o n enzyme a c t i v i t y a f t e r d i a l y s i s i n b u f f e r C. 6. Assay Column e l u e n t s were a s sayed f o r s p e c i f i c e n d o n u c l e o l y t i c a c t i v i t y u s i ng A DNA (a g i f t of Dr. P. Denn i s ) as the s u b s t r a t e s i m i l a r to the a s say method d e s c r i b e d by Sharp et al ( 1973 ) . A l i q u o t s o f column f r a c t i o n s were i n c u b a t e d i n 20 y l r e a c t i o n m i x t u r e c o n t a i n i n g 1.5 yg X DNA and b u f f e r D (6 mM T r i s - H C l , - 13 -pH 7 .9 , 6 mM MgCl 2m 6 mM 2 - m e r c a p t o e t h a n o l ) at 37°C f o r 2 h. F i v e . u l o f s top mix A (40% w/v s u c r o s e , 0.025% w/v bromophenol b l u e , 0.025 M EDTA, pH 7.4) was added. The m i x t u r e was l o aded onto a 1 .4% ' ' agarose ( B i o R a d , e l e c t r o p h o r e t i c grade) h o r i z o n t a l s l a b ge l 100 mm long and a p p r o x i m a t e l y 3 mm t h i c k i n TBE b u f f e r (90 mM T r i s - b o r a t e pH 8 .3 , 90 mM b o r i c a c i d , 25 mM EDTA) (Peacock & Dingman, 1967) c o n t a i n i n g 1 yg/ml e t h i d i u m bromide and e l e c t r o p h o r e s e d a t 400 V f o r 1 h. The ge l was then p h o t o -graphed under u l t r a v i o l e t l i g h t on P o l a r o i d f i l m . ( t y p e 57) u s i ng an orange f i l t e r . One u n i t o f r e s t r i c t i o n enzyme a c t i v i t y was d e f i n e d as the amount o f enzyme r e q u i r e d to c o m p l e t e l y d i g e s t 1 yg of X DNA a t 37°C i n i h. A l l i s o l a t i o n and p u r i f i c a t i o n p r o cedu re s were r e p e a t e d at l e a s t once to check f o r r e p r o d u c i b i l i t y . C h a r a c t e r i z a t i o n o f r e s t r i c t i o n enzymes 1. D i g e s t i o n of d o u b l e - s t r a n d e d b a c t e r i o p h a g e DNA I n i t i a l c h a r a c t e r i z a t i o n of the p u r i f i e d r e s t r i c t i o n enzymes i n v o l v e d d i g e s t i o n o f $X174am3 r e p l i c a t i v e form DNA ($X RF DNA; p r e p a r a t i o n w i l l be d e s c r i b e d l a t e r ) . Compar i son o f the e l e t r o p h o r e t i c p a t t e r n s of the unknown r e s t r i c t i o n d i g e s t i o n p r o d u c t s w i t h computer p r e d i c t e d f ragment p a t t e r n s p e r m i t t e d i d e n t i f i c a t i o n of the r e c o g n i t i o n sequence. Double d i g e s t s c o n f i r m e d i s o s c h i z o m e r a c t i v i t y when no a d d i t i o n a l - 14 -DNA f ragments were g ene r a t ed i n the p re sence o f both the new enzyme and one of known s p e c i f i c i t y . I f the enzyme were r e c o g n i z i n g a new sequence then doub le d i g e s t i o n p a t t e r n s c o u l d be used to l o c a t e d some c l e a vage s i t e s on the genome. D i g e s t i o n s o f $X RF DNA (2 yg) were c a r r i e d out i n 20 y l r e a c t i o n volumes i n the p re sence o f b u f f e r D a t 37°C. The t ime of i n c u b a t i o n v a r i e d depend ing on the amount of enzymes used. F i v e y l o f s top mix A was added to the r e a c t i o n m i x t u r e s wh ich were then l oaded onto a 5% a c r y l a m i d e (Eastman) v e r t i c a l s l a b gel 1.5 x 200 x 200 mm; made from a d e i o n i z e d s t o c k s o l u t i o n of 29% a c r y l a m i d e , 1% b i s a c r y l amide ( i n H^0, w/v) i n TBE b u f f e r and e l e c t r o -phoresed a t 100 V f o r 4 h. A f t e r s t a i n i n g f o r 0.5 h i n an aqueous s o l u t i o n of e t h i d i u m bromide (1 yg/ml) the gel was photographed as p r e v i o u s l y d e s c r i b e d i n the a s say p r o c e d u r e . Other v i r a l DNAs were s u b s t i t u t e d i f the new enzyme d i d not cu t $X RF DNA. 2. 2-D e l e c t r o p h o r e s i s and homochromatography Seqlienc i ng . o f 5 1 -1 a b e l l ed o l i godeoxyr i bonuc 1 eo t i des by two d i m e n s i o n a l (2-D) f r a c t u r a t i o n ( a l s o r e f e r r e d to as wander ing spot a n a l y s i s ) by e l e c t r o p h o r e s i s on a c e l l u l o s e a c e t a t e s t r i p a t pH 3.5 f o l l o w e d by homochromatography on a D E A E - c e l l ul ose t h i n l a y e r p l a t e (Sanger e't al, 1 973) has been adapted f o r d e t e r m i n i n g the r e c o g n i t i o n sequence o f - 15 -enzymes t h a t c l e a v e w i t h i n the s i t e to g i v e f r agment s w i t h 5' e x t e n s i o n s ( Robe r t s et al, 1977) . a) ['M Pl-ATP exchange DNA (3.5 ug) d i g e s t i o n was c a r r i e d out a t pH 7.5 under s i m i l a r c o n d i t i o n s as d e s c r i b e d above and the r e a c t i o n was s topped by h e a t i n g at 100°C f o r 3 m in . The 5 1 - p h o s p h o r y l ends gene ra ted by r e s t r i c t i o n c l e a v a g e s were l a b e l l e d by the exchange method o f Be rkne r and Fo l k ( 1977 ) . The exchange r e a c t i o n m i x t u r e c o n t a i n e d the heat i n a c t i v a t e d DNA d i g e s t , [ y 3 2 P ] - A T P (30 pmole, 3000 Ci/mmole, New Eng land N u c l e a r ) , 10 yM ATP, 800 yM ADP, 4.5 mM d i t h i o t h r e i t o 1 , 18 mM M g C l 2 , 45 mM KC1, 25 mM T r i s - H C l pH 7.4 i n a volume of 50 y l . P o l y n u c l e o t i d e k i n a s e (5 u n i t s , New Eng land B i o l a b ) was added and the r e a c t i o n m i x t u r e i n c u b a t e d a t 37°C f o r 15 min a f t e r wh ich 1 y l a l i q u o t was removed and an equal volume o f water s a t u r a t e d phenol was added. The 1 y l a l i q u o t t h a t was removed was d i l u t e d 1 0 x w i t h d i s t i l l e d water and used f o r e s t i m a t i n g the e x t e n t o f l a b e l -l i n g u s i n g paper ch romatog raphy . One y l o f a 10.x d i l u t e d a l i q u o t f rom the exchange r e a c t i o n and 50-100 nmoles o f c a r r i e r ATP were s p o t t e d on DEAE - ce l 1u l o s e (DE 81 , Whatman) paper s t r i p s (2 cm wide) and de s cend i ng chromatography w i t h a 0.35 M ammonium fo rmate pH 8.0 was used to s e p a r a t e r e s t r i c t i o n f ragments f rom ATP. The amount o f u n r e a c t e d 32 [y P]-ATP can be counted ( s c i h t i l l a t i on) i n the uv -abso rb ing ATP - 16 -spot and compared w i t h the P - l a b e l l e d p o l y n u c l e o t i d e s a t the o r i g i n . b) Purification of labelled fragments The phenol i n the r e a c t i o n m i x t u r e was removed by 2x e x t r a c t i o n w i t h e t h e r . Excess e t h e r r e m a i n i n g i n the aqueous phase was e v a p o r a t e d u s i ng ah a i r j e t . :The aqueous s o l u t i o n of l a b e l l e d f ragment s was a p p l i e d to a 1 ml ( a p p r o x i m a t e l y 1.5 x 15 mm) column o f Sephadex G-100 (Pha rmac i a ) p r e -e q u i l i b r a t e d i n a b u f f e r c o n t a i n i n g 0.05 mM EDTA and 2.0 mM T r i s - H C l pH 7.5 and e l u t e d w i t h the same b u f f e r . E l u t i o n p r o f i l e s were m o n i t o r e d w i t h a handhe ld g e i g e r c o u n t e r . The l a b e l l e d r e s t r i c t i o n f ragments e l u t e d i n the v o i d volume were c o l l e c t e d and l y o p h i l i z e d . c) Digestion with P^ enzyme D i g e s t i o n of the f ragments was c a r r i e d out i n 10 y l volumes at room tempe ra tu re f o r 10 min i n the p re sence o f 1 yg o f heat dena tu red c a l f thymus DNA, 10 mM T r i s - H C l pH 7.5 and 5 y l o f 2 un i t s /1 000 ml P-|enzyme (PL Bi ochemi c a l s) * . The r e a c t i o n was s topped w i t h 1 y l o f 0.1 M EDTA f o l l o w e d by h e a t i n g a t 100°C f o r 3 min . These r e a c t i o n c o n d i t i o n s gave a hete rogeneous m i x t u r e of f ragments r a n g i n g from monomers to o l i g o m e r s of v a r i o u s l e n g t h s s u i t a b l e f o r 2-D f r a c t i o n a t i o n ( C R . A s t e l l , p e r s ona l c o m m u n i c a t i o n ) . * P| enzyme ( f rom Penicillium citrinum) was o b t a i n e d i n the l y o p h i l i z e d form w i t h a p p r o x i m a t e l y 600 un i t s/mg and was d i s s o l v e d i n 50% g l y c e r o l , 10 mM T r i s - H C l pH 7.5..and s t o r e d a t - 20°C . P] i s an endonuc lea se t h a t c l e a v e s randomly . - 17 -d) Electrophoresis on cellulose acetate Pi d i g e s t e d samples were a p p l i e d to 3mm c e l l u l o s e a c e t a t e s t r i p s ( S c h l e i c h e r & S c h u e l l ) p r e - w e t t e d i n 7 M u r e a , p y r i d i n e - a c e t a t e (5% v/v) pH 3.5 and e l e c t r o p h o r e s e d i n the same b u f f e r f o r 1.5 h u n t i l the b l ue and y e l l o w dyes ( s p o t t e d 5.mm from the edges) were about 100-150 mm a p a r t . e) Homochromatography The l a b e l l e d m a t e r i a l s on the c e l l u l o s e a c e t a t e s t r i p was l o c a t e d w i t h a hand h e l d g e i g e r c o u n t e r and t r a n s f e r r e d by the Sou the rn method (1974) onto a 400 x 200 mm D E A E - c e l 1 u l o s e t h i n l a y e r shee t (DEAE/HR-2/15, Mache rey -Nage l ) mounted on a g l a s s p l a t e . Pads of 3MM paper (40 x 200 mm) p r e - s o a k e d i n water were p l a c e d ove r the edges of the c e l l u l o s e a c e t a t e s t r i p . . The wa te r was used to c a r r y the n u c l e o t i d e s and the dyes to the DEAE shee t where i t i s he l d by i o n i c f o r c e s a t the p o i n t of c o n t a c t as shown i n F i g . 1. The DEAE p l a t e was r i n s e d i n e t h a n o l to remove urea t h a t was t r a n s f e r r e d , d r i e d a t 70°C then sp rayed l i g h t l y w i t h d i s t i l l e d r^O a t the bottom h a l f of the s h e e t . Homo-chromotography was then c a r r i e d out i n the p re sence of 2% p a r t i a l l y h y d r o l y z e d y e a s t RNA (1 to 14 n u c l e o t i d e s i n l e n g t h ) i n 7 M urea (homomix V, Jay et al, 1974) a t 70°C u n t i l the b lue dye had m i g r a t e d to 20-30 mm from the top o f the p l a t e (5-6 h ) . The 2-D f r a c t i o n a t i o n map was o b t a i n e d by e xpo s i n g a sheet o f Kodak NS-5T X - r a y f i l m f o r an a p p r o p r i a t e l e n g t h of t ime depend ing on the amount o f l a b e l p r e s e n t . - 18 -F i g 1 Diagram showing the s e t up f o r t r a n s f e r r i n g m a t e r i a l f rom c e l l u l o s e a c e t a t e s t r i p to D E A E - c e l 1 u l o s e s h e e t . glass plate 2ml pipette = ^ o * DEAE-cellulose w \ th in layer sheet V X O MM n a n n y ' 3 MM paper ce l lu lo se actate s t r i p f ) Determination of 5'-terminus The 5 ' - t e r m i n a l base of the r e s t r i c t i o n c l e a v a g e was de te rm ined by paper e l e c t r o p h o r e s i s ( B a r r e l l , 1971 ) . The l a b e l l e d o l i g o m e r i c f r agment s (20,000 to 50,000 cpm) were d i g e s t e d to mononuc l eo t i de s w i t h an exce s s o f P-j enzyme (2 u n i t s ) u s i n g the c o n d i t i o n s d e s c r i b e d p r e v i o u s l y and the m i x t u r e was a p p l i e d onto a p i e c e of 23 x 35 cm 3MM (Whatman) paper a l o n g s i d e samples o f the f o u r d e o x y r i b o -n u c l e o t i d e monophosphates s t a n d a r d s . A spo t o f homomix dye c o n s i s t i n g o f Xy lene cyano l ( b l u e ) , a c i d f u c h s i n ( r ed ) and methy l orange ( y e l l o w ) was added as markers ( S ange r , 1965 ) . The paper was impregnated ( e x c e p t a t the o r i g i n ) w i t h p y r i d i n e - a c e t a t e , 5% ( v / v ) , pH 3.5 and e l e c t r o p h o r e s e d a t 2000 V f o r 2 h i n the same b u f f e r . M o n o n u c l e o t i d e spo t s - 19 -f rom the P-j d i g e s t i o n were e x c i s e d and counted i n s c i n t i l -l a t i o n v i a l s c o n t a i n i n g 5 ml O m n i f l u o r (New Eng land N u c l e a r ) . 3. D e t e r m i n a t i o n of r e s t r i c t i v e enzyme c l e a v a g e s i t e s The 2-D f r a c t i o n a t i o n method a l l o w s d e t e r m i n a t i o n o f t h e r e c o g n i t i o n sequence o n l y i f the r e s t r i c t i o n c l e a v a g e o c c u r r e d w i t h i n the s i t e o f r e c o g n i t i o n and even when t h i s c o n d i t i o n i s met, a m b i g u i t y may s t i l l a r i s e and a n a l y s i s o f the 3' t e r m i n a l sequence i s o f t e n r e q u i r e d . For c l e a v a g e s o u t s i d e o f the r e c o g n i t i o n s i t e , t h i s method i s not a p p l i c a b l e . The method o f Brown and Smith (1976) u t i l i z e s the r a p i d p l u s minus s equenc i ng t e c h n i q u e by pr imed s y n t h e s i s w i t h DNA po lymerase I (Sanger & C o u l s o n , 1975) to l o c a t e the c u t s i t e s and a l l o w d e d u c t i o n of r e c o g n i t i o n sequence i r r e s p e c -t i v e o f c l e a v a g e b e h a v i o r . D e t e r m i n a t i o n of the c l e a v a g e s i t e i s d e t a i l e d i n Brown and Smith (1977a) and the p l u s - m i n u s s equenc i ng method used i n t h i s t h e s i s i s i d e n t i c a l to t h a t g i v en by Brown and Smith ( 1 977b) e x c e p t as f o l l o w s . V i r a l ( + ) <J>X174am3 DNA was used as t e m p l a t e f o r the DNA po lymerase I c a t a l y z e d e x t e n s i o n ; E. coli DNA po lymerase I and bacter iophage T4 po lymerase were p r epa red by C.R. A s t e l l ; [ a 3 2 P ] - d A T P ( a p p r o x i m a t e l y 70 pmoles , 300-350 Ci/mmoles f rom New England N u c l e a r ) per e x t e n s i o n r e a c t i o n was u sed. A u t o r a d i o g r a p h s were o b t a i n e d . by e xpo s i n g Kodak NS-5T X - r a y f i l m f o r 2-7 days . - 20 -4. C leavage mapping by n e a r e s t ne i ghbou r a n a l y s i s . C leavage maps o f enzymes w i t h nove l s p e c i f i c i t i e s were o b t a i n e d by p u l s e l a b e l l i n g r e s t r i c t i o n f r agment s w i t h DNA po lymerase (Jeppesen et al, 1976) . a) Preparation of restriction fragments $X RF DNA (100 yg) was d i g e s t e d w i t h one u n i t of enzyme, per ug DNA i n 650 yl volumes c o n t a i n i n g b u f f e r D f o r 12 h a t 37°C. The m i x t u r e was t r e a t e d w i t h 650 y l phenol wh ich was s u b s e q u e n t l y removed by 4 x e t h e r e x t r a c t i o n . Excess e t h e r was blown o f f w i t h a j e t o f a i r . The d i g e s t e d DNA was made o . l M i n sodium a c e t a t e and p r e c i p i t a t e d w i t h 2 volumes o f 95% e t h a n o l . The p r e c i p i t a t e was d i s s o l v e d i n 100 y l o f 50 mM KC1 and s top mix A (25 y l ) was added p r i o r to l o a d i n g onto a p r e p a r a t i v e 5% a c r y l a m i d e ge l (3 .x 1.70 x 400 mm) i n TBE b u f f e r and e l e c t r o p h o r e s e d a t 80 V f o r 18.5 h. The ge l was s t a i n e d as d e s c r i b e d i n s e c t i o n 1 o f c h a r a c t e r i z a t i o n p r o c e d u r e s . DNA f r agment s were e x t r a c t e d by e l e c t r o e l i u t i o r i u s i n g 10 ml (8mm d i a m e t e r ) p l a s t i c p i p e t t e s ( F a l c o n ) c u t down to 180 mm l e n g t h s p lugged w i t h g l a s s wool a t the t a p e r e d t i p ( G a l i b e r t et al, 1974 ) . A p i e c e of d i a l y s i s t u b i n g ( a p p r o x -i m a t e l y 5 mm d i a m e t e r ) , f i t t e d to the p i p e t t e t i p a t one end and doub l y k n o t t e d a t the o t h e r , was used to r e t a i n the e l u t e d DNA f r a g m e n t s . Gel bands were e x c i s e d and i n d i v i d u a l l y - 21 -e x t r u d e d f rom 10 ml d i s p o s a b l e s y r i n g e s (no n e e d l e ) i n t o s e p a r a t e e l e c t r o p h o r e s i s p i p e t t e s c o n t a i n i n g EEB b u f f e r (20 mM T r i s - a c e t a t e pH 8 .0 , 1mM EDTA) and e l e c t r o p h o r e s e d i n the same b u f f e r a t 2 mamps per tube f o r a p p r o x i m a t e l y 16 h. The s o l u t i o n c o n t a i n i n g the DNA was removed f rom the d i a l y s i s t u b i n g , e t h a n o l p r e c i p i t a t e d as p r e v i o u s l y d e s c r i b e d , d i s s o l v e d i n 100 ul d i s t i l l e d H 2 0 and s t o r e d a t - 2 0°C . b) Mapping of DNA fragments by "nearest neighbour" analysis The p u r i f i e d f ragment s s e r ved as p r i m e r s f o r p u l s e l a b e l l i n g by E. coli DNA po lymerase on v i r a l (+) $X DNA tempi a t e . Template DNA (0.125 yg i n 0.25 y l s o l u t i o n ) was mixed w i t h 5 y l of the above p r epa red f r agment s o l u t i o n i n a s i l i c o n i z e d tube and heated a t 100°C f o r 3 min i n a s e a l e d c a p i l l a r y t u b e . The p r i m e r and t e m p l a t e were a l l o w e d to anneal a t 65°C f o r 2.5 h i n the p re sence of b u f f e r E (50 mM NaCI, 6.6 mM M g C l 2 > 6.6 mM 2 - m e r c a p t o e t h a n o l , 6.6 mM T r i s - H C l pH 7 . 4 ) . A f t e r c o o l i n g , the s o l u t i o n was i n c u b a t e d a t 4°C f o r 1 min w t i h 0.9 u n i t s (1 y l ) o f DNA po lymerase (nach K lenow, B o e h r i n g e r Mannheim) i n the p re sence of [ a 3 2 P ] - d A T P ( a p p r o x i -mate l y 2-3 pmole, 341.9 C i /mmo le ) , 50 yM dCTP, dGTP, dTTP i n a f i n a l volume o f 16 y l . Exces s dATP (2.5 y l of 0.5 mM) was added to chase the r a d i o a c t i v e n u c l e o t i d e . R e s t r i c t i o n enzyme o r i g i n a l l y used f o r p r e p a r i n g the p r i m e r s was added (0.25 y l , about 0.05 u n i t s , per r e a c t i o n ) to r e g e n e r a t e the f r agment s and the m i x t u r e was i n c u b a t e d w i t h an a d d i t i o n a l - 22 -0.25 y1! o f 10 x b u f f e r E f o r 30 min a t 37°C. F i v e y l o f s top mix A were added to the s o l u t i o n s p r i o r to l o a d i n g onto a 5% a c r y l a m i d e gel (1.5 x 170 x 400 mm) i n TBA b u f f e r and e l e c t r o -phoresed a t 100 V f o r 16 h. E x a m i n a t i o n of the a u t o r a d i o g r a p h s hou l d a l l o w i d e n t i f i c a t i o n of l a b e l l e d bands found a d j a c e n t to the p r i m e r f r a gmen t . P r e p a r a t i o n o f <j>X174am3 DNA DNA was p repa red from a b a c t e r i o p h a g e s t o c k of l y s i s d e f e c t i v e $X174am3 ( f i r s t i s o l a t e d by H u t c h i s o n & S i n s h e i m e r , 1966) , a g i f t o f C.A. Hu t ch i s on I I I , by the f o l l o w i n g method ( H u t c h i s o n , C.A. I l l , p e r s o n a l c o m m u n i c a t i o n ) . 1 . Phage growth N o n - p e r m i s s i v e hos t E. coli C was grown to 3 x 1 0 8 c e l l s per ml i n 15 I B b r o t h (pH 7.0) c o n t a i n i n g 1% (w/v) t r y p t o n e ( D i f c o ) , 0.8% (w/v) N a C l , 0.1% (w/v) d e x t r o s e i n a 30 j f e r m e n -t o r ( F e r m e n t a t i o n Des ign) i n the p re sence of 1 mM C a C ^ ( r e q u i r e d f o r the a t t achment of the b a c t e r i o p h a g e to the ho s t ) a t 37°C, a e r a t e d a t a f l o w r a t e s e t t i n g of a p p r o x i m a t e l y 18 and s t i r r e d a t 200 rpm. The c e l l s were i n f e c t e d w i t h <£>X174am3 phage a t a m u l t i p l i c i t y o f i n f e c t i o n of 3 -5 . V i r a l (+) DNA ( s i n g l e - s t r a n d e d ) r e q u i r e s phage p r o t e i n f o r s y n t h e s i s but the r e p l i c a t i v e form (RF) of the DNA ( d o u b l e - s t r a n d e d ) i s s y n t h e s i z e d by hos t gene, p r o d u c t s ( E i s e n b e r g et al, 1 976 ) . Thus i n the p r e p a r a t i o n o f RF DNA, c h l o r a m p h e n i c o l (450 mg d i s s o l v e d i n 100 ml B b r o t h ) was added 10 min a f t e r i n f e c t i o n to i n h i b i t - 23 -( + ) - s t r a n d f o r m a t i o n by b l o c k i n g de novo p r o t e i n s y n t h e s i s . The un l y s ed ho s t c e l l s were h a r v e s t e d i n a S h a r p i e s c e n t r i f u g e 2.5 h a f t e r i n f e c t i o n and f r o z e n a t - 20°C . 2. P u r i f i c a t i o n o f RF DNA The c e l l p e l l e t was re suspended i n 75 ml o f 25% s u c r o s e (w/v) 0.05 M T r i s - H C l pH 8.0 a t room tempe ra tu re and i n c u b a t e d w i t h 75 mg ly sozyme ( Se i kagaku ) f o r 5 min a t 0 -4°C . EDTA (30 ml 0.25 M pH 8, 5 min i n c u b a t i o n a t 0-4°C) was added to f a c i l i t a t e l y s i s . 120 ml of 10% T r i t o n X-100 ( S i g m a ) , 50 mM T r i s - H C l pH 8, 60 mM EDTA was added p r i o r to c e n t r i f u g a t i o n a t 20,000 rpm i n Beckman type 35 r o t o r f o r 30 m in . P o l y -e t h l y l e n e g l y c o l (100 ml of a 30% w/v s o l u t i o n i n 1.5 M NaCI) was added to the s u p e r n a t a n t ( a p p r o x i m a t e l y 200 ml) to p r e -c i p i t a t e the DNA. The DNA p e l l e t f rom c e n t r i fuga^tion ( 8 -10 ,000 x g, 15 min) was re su spended i n 120 ml 100 mM T r i s -HCl pH 8, 10 mM EDTA and p u r i f i e d by 2 c o n s e c u t i v e runs of equi l ibr ium g r a d i e n t centr i f ugati on ( 30,5 00 rpm i n Beckman type 35 r o t o r , 65 h) w i t h an average CsC l ( S i gma, s p e c t r a l g rade) d e n s i t y of 1.6 g/ml i n the p re sence o f e t h i d i u m bromide (50-100 ug/m l ) . The l ower band c o n t a i n i n g the phage DNA was e x t r a c t e d w i t h i s o p r o p a n o l to remove e t h i d i u m b romide , d i a l y z e d a g a i n s t 20 mM T r i s - H C l pH 7 . 5 , 2mM EDTA, e x t r a c t e d w t i h H 2 0 - s a t u r a t e d phenol :CHC1 3 ( 1 : 1 , v / v ) , d i a l y z e d e x t e n s -i v e l y a g a i n s t 5mM T r i s - H C l pH 7 .5 , 0.1 mM EDTA and kept f r o z e n at - 20°C . The p r e p a r a t i o n y i e l d e d about 4 mg of phage RF DNA - 24 -as de te rm ined by assuming t h a t an abso rbance o f 44.7 yg RF DNA/ml a t 260 nm wave leng th i s 1.0 ( S i n s h e i m e r , 1966 ) . R e s u l t s from ge l e l e c t r o p h o r e s i s i n 1% agarose showed a s i n g l e band o f RF DNA t h a t was f r e e f rom c o n t a m i n a t i n g v i r a l s i n g e l - s t r a n d e d and host DNA. 3. P u r i f i c a t i o n of v i r a l (+) s t r a n d DNA The c e l l p e l l e t was re su spended i n 35 ml 0.1 M b o r a t e ( sod ium s a l t , pH 9 r e q u i r e d to de tach phage f rom host membranes) and i n c u b a t e d i n 20 mg ly sozyme a t room t empe ra tu re f o r 10 m i n . F o l l o w i n g the a d d i t i o n of 6 ml 0.1 M EDTA pH 7.4, the s o l u t i o n was i n c u b a t e d f o r a f u r t h e r 60 min u n t i l ve ry v i s c o u s . The v i s c o s i t y was reduced by s h o r t s o n i c a t i o n . C e l l u l a r d e b r i s was removed by s p i n n i n g a t 10,000 x g, 15 min and the phage p a r t i c l e s ( o p a l e s c e n t ) i n the s u p e r n a t a n t were banded by l i n e a r g r a d i e n t c e n t r i f u g a t i o n (30,000 rpm i n Beckman type 35 r o t o r , 24 h) i n CsCl (average d e n s i t y o f 1.38 g/m l ) , 0.1 M b o r a t e pH 9. The band c o n t a i n i n g the phage was removed and f u r t h e r p u r i f i e d by c e n t r i f u g a t i o n th rough 10% s u c r o s e i n t o a CsCl s t e p w i s e g r a d i e n t w i t h s p e c i f i c d e n s i t i e s o f 1.7, 1.5 and 1.3 g/ml r e s p e c t i v e l y . The band of h i g h l y p u r i f i e d phage was then d i a l y z e d i n 0.05 M b o r a t e pH 9 and e x t r a c t e d w i t h p h e n o l . Excess phenol r e m a i n -i n g i n the aqueous phase was removed by 4-6 x e t h e r e x t r a c t i o n and the DNA s o l u t i o n was c o n c e n t r a t e d by i s o b u t a n o l e x t r a c t i o n , d i a l y z e d a g a i n s t 5 mM T r i s - H C l pH 7 .5 , 1 mM EDTA and kept - 25 -f r o z e n a t - 2 0°C . Assuming 36 y g / m l / A 2 6 0 ( S i n s h e i m e r , 1966 ) , about 3 mg o f v i r a l (+) s t r a n d DNA w i t h s u f f i c i e n t p u r i t y f o r DNA sequenc i ng a p p l i c a t i o n was o b t a i n e d . - 26 -RESULTS The b a c t e r i a The a n a e r o b i c b a c t e r i a used t h i s t h e s i s were i s o l a t e d f rom the human o r a l c a v i t y . The s p e c i e s t h a t was most p r o -d u c t i v e in r e s t r i c t i o n a c t i v i t y was the Fusobaoterium nuoleatum a g r am-nega t i v e o b l i g a t e anaerobe t h a t was e a s i l y i d e n t i f i e d by i t s t y p i c a l need l e - s haped morphology wh i ch d i s i n t e g r a t e d i n t o sma l l p a r t i c l e s when s o n i c a t e d . F u r t h e r c h a r a c t e r i z a t i o n by gas chromatography o f e t h e r e x t r a c t s f rom the a c i d i f i e d c u l t u r e s u p e r n a t a n t showed an e l u t i o n p r o f i l e w i t h a major b u t y r i c a c i d peak t h a t i s c h a r a c t e r i s t i c o f the Fusobaoterium genus(Moore et al, 1972 ) . The s p e c i e s , i F . nuoleatum, produces i n d o l e as w e l l . These compounds t o g e t h e r w i t h gaseous p r o d u c t s c o n s t i t u t e the f o u l odor t h a t d i f f e r e n t i a t e s the F. nuoleatum f rom o t h e r o r a l o rgan i sms w i t h s i m i l a r morpho-l o g y . The F. nuoleatum i s o l a t e d from v a r i o u s i n d i v i d u a l s were d e s i g n a t e d as d i f f e r e n t s t r a i n s a l t h o u g h they were s i m i l a r i n p r o p e r t i e s ( M c B r i d e , B .C. , pe r s ona l c o m m u n i c a t i o n ) . r'Enu D 1. P u r i f i c a t i o n of Fnu DI, Fnu DII and Fnu D i l i Three s i t e s p e c i f i c d e o x y r i b o e n d o n u c l e a s e s , Fnu D I , Fnu DII and Fnu D i l i , were p u r i f i e d from Fusobaoterium - 27 -nucleaturn D. A l l the r e s t r i c t i o n enzymes examined i n t h i s t h e s i s s t a r t e d to e l u t e f rom B i o g e l A 0.5 m a t around the same f r a c t i o n s as seen i n F i g . 2. The p re sence of a t l e a s t two d i f f e r e n t r e s t r i c t i o n a c t i v i t i e s i n F. nucleaturn D was appa ren t a f t e r the B i o g e l column ( F i g . 2 ) . F r a c t i o n s t h a t gave d i s c r e t e bands on ge l e l e c t r o p h o r e s i s when i n c u b a t e d w i t h A DNA c o n t a i n e d e n d o n u c l e o l y t i c a c t i v i t y and were p o o l e d , d i a l y z e d and a p p l i e d to a p h o s p h o c e l 1 u l o s e co lumn. Fnu DI and Fnu DII c o - e l u t e d w i t h a p p r o x i m a t e l y 0.06 M KC1 , and Fnu D i l i w i t h 0.35 M KC1 ( F i g . 3 ) . Fnu D i l i was c o n c e n t r a t e d a t l e a s t f i v e f o l d on a second p h o s p h o c e l 1 u l o s e column by s t e p w i s e e l u t i o n w i t h 1 M KC1 w i t h l i t t l e l o s s i n r e s t r i c t i o n a c t i v i t y . The p u r i f i e d Fnu D i l i c o n t a i n e d no n o n - s p e c i f i c n u c l e a s e c o n t a m i n a t i o n as judged from the absence o f chan.ge i n band p a t t e r n when X DNA was d i g e s t e d w i t h a l a r g e exce s s o f enzyme . Fnu DI and Fnu DII were s e p a r a t e d on a DEAE-Sephadex column by e l u t m g w i t h a l i n e a r g r a d i e n t of KC1 (0 to 0.3 M). Fnu DI e l u t e d a t a p p r o x i m a t e l y 0.05 M KC1 f r e e o f Fnu DII and n o n - s p e c i f i c n u c l e a s e a c t i v i t y , and Fnu.DII a t 0.1 M KC1 w i t h low c o n c e n t r a t i o n s of c o n t a m i n a t i n g DI p r e s e n t ( F i g . 4 ) . More r e c e n t e x p e r i m e n t s showed t h a t b e t t e r s e p a r a t i o n was e f f e c t e d on a DEAE-Sephacel u s i n g a 0-0.2 M l i n e a r g r a d i e n t o f KC1 and Fnu DII was p u r i f i e d f r e e o f c o n t a m i n a t i n g Fnu D I . A l l r e s t r i c t i o n enzymes r e p o r t e d i n t h i s t h e s i s were - 28 -s t a b l e over 12 months when s t o r e d i n 50% g l y c e r o l a t - 2 0°C . Over 1 000..units o f each of the Fnu D enzymes were o b t a i n e d f rom 8 g o f f r o z e n c e l l s . O v e r a l l r e c o v e r i e s were d i f f i c u l t to de te rm ine because of the i n t e r f e r e n c e o f n o n - s p e c i f i c n u c l e a s e s i n c rude e x t r a c t a s s a y s . - 29 -F i g . 2. Assay o f f r a c t i o n s f rom B i o g e l A 0.5 m column of Fnu D r e s t r i c t i o n enzymes a n a l y z e d by . gel e l e c t r o p h o r e s i s . A l i q u o t s (5 y l ) o f column . f r a c t i o n s (5 ml) were a s sayed as d e s c r i b e d i n the methods and m a t e r i a l s and 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 on a 1 .4% agarose ge l c o n t a i n i n g 1 yg/ml e t h i d i u m bromide and photographed under u l t r a v i o l e t l i g h t . Assay r e s u l t s of a l l column f r a c t i o n s i n subsequent f i g u r e s were o b t a i n e d i n the same manner. fraction no. - 30 -F i g . 3. Assay o f f r a c t i o n s from p h o s p h o c e l 1 u l o s e column o f Fnu D r e s t r i c t i o n enzymes by gel e l e c t r o -p h o r e s i s o f d i g e s t e d X DNA. Fnu DI & II fraction no. 62 64 66 68 70 74 Fnu Dill - 31 -F i g . 4. Assay o f f r a c t i o n s from DEAE-Sephadex A-50 column o f Fnu DI and Fnu DII by ;gel e l e c t r o p h o r e s i s of d i g e s t e d A DNA Fnu DI fraction no. 31 33 35 37 39 41 Fnu DII - 32 -2. C h a r a c t e r i z a t i o n of Fnu DI, Fnu DII and Fnu D i l i a) Effect of salt concentration A l l t h r e e enzymes were i n h i b i t e d by NaCl c o n c e n t r a t i o n s above 300 mM i n b u f f e r D. Fnu DI appeared to be most a c t i v e i n the absence o f NaCl . The optimum NaCl c o n c e n t r a t i o n f o r Fnu DII was 50-150 mM and f o r Fnu D i l i was 50 mM N a C l . b) Stability A n a l y s i s by e l e c t r o p h o r e s i s on 1.4% agarose g e l s of 1-8 yg of A DNA i n c u b a t e d f o r 12 h w i t h 1 u n i t o f Fnu DII i n b u f f e r D c o n t a i n i n g 100 mM NaCl showed t h a t the enzyme was a c t i v e f o r up to 8 h a t 37°C. c) Double digestion analysis of Fnu DI and Fnu Dili The absence of a d d i t i o n a l f r agment s f rom doub le d i g e s t -i on e x p e r i m e n t s u s i n g $X RF DNA and S13 RF DNA showed t h a t Fnu DI and Fnu..;DIII were i so sch i zomers o f Hae I I I ( f r om Haemophilus aegyptius) and Hha I ( f rom Haemophilus haemoly-ticus) r e s p e c t i v e l y . Thus Fnu DI r e c o g n i z e s the sequence 5 ' - G G C C - 3 ' , and Fnu D i l i r e c o g n i z e 5 ' - G C G C - 3 ' . The c l e a v a g e s p e c i f i c i t i e s o f these enzymes were f u r t h e r c h a r a c t e r i z e d by the Brown and Smith method as d e s c r i b e d i n the methods and m a t e r i a l s s e c t i o n . d) Sequencing cleavage sites of Fnu DI and Fnu Dili From known r e s t r i c t i o n enzyme c l e a v a g e maps o f $X DNA Hind II f ragment 8* ( a b b r e v i a t e d R8) was the most s u i t a b l e *A11 p r i m e r f ragment s used f o r s equenc i ng were o b t a i n e d from r e s t r i c t i o n enzyme d i g e s t e d $X RF DNA p repa red as d e s c r i b e d i n methods and m a t e r i a l s . - 33 -p r i m e r f o r mapping an a d j a c e n t Fnu DI c l e a v a g e s i t e . The p r i m e r , annea led to $X v i r a l (+) DNA t e m p l a t e i n the p re sence o f DNA po lymerase I, [ a 3 2 P ] - d A T P , u n l a b e l l e d dGTP, dCTP and dTTP gene ra ted a p u l s e l a b e l l e d copy s t r a n d . Un reac ted mononuc leo-t i d e s were removed by chromatography on G-100 column (see s e c t i o n on p u r i f i c a t i o n o f l a b e l l e d f r agment s i n methods and m a t e r i a l s ) . A l i q u o t s o f the e x t e n s i o n p r o d u c t were used f o r subsequent i n c u b a t i o n w i t h " p l u s - m i n u s " m i x t u r e s . The m i x t u r e s were d i g e s t e d w i t h Hind I I to remove the p r i m e r f ragment and the p r o d u c t s used to produce a ge l e l e c t r o p h o r e s i s sequence p a t t e r n o f the sequence a d j a c e n t to f ragment R8 ( F i g . 5 ) . To de te rm ine the c l e a v a g e s i t e s f o r Fnu DI, a d d i t i o n a l r e a c t i o n m i x t u r e s were run ( c h a n n e l s F and F' i n F i g . 5) a l o n g s i d e the p l u s and minus s equenc i ng p a t t e r n s . The F channe l c o n t a i n e d a l a b e l l e d copy s t r a n d gene ra ted by d i g e s t i n g an a l i q u o t of the e x t e n s i o n p r oduc t w i t h Fnu DI and datum enzyme, Hind I I . ".The l e n g t h o f the r e s u l t a n t f r agment ex tended f rom the 5 ' -homogeneous end of the datum c u t to the f i r s t c l e a v a g e s i t e o f Fnu DI. T h i s a l l o w e d d e t e r m i n a t i o n o f the p o i n t of c l e a v a g e on the l a b e l l e d s t r a n d . C leavage by r e s t r i c t i o n endonuc l ea se s c o u l d gene ra te f ragment s w i t h e i t h e r 5 ' - or 3 ' - e x t e n s i o n s or f l u s h ends . To de te rm ine the mode of c l e a v a g e i n the s t r a n d complementary to the l a b e l l e d s t r a n d , T4 DNA po lymerase and d e o x y n u c l e o s i d e t r i p h o s p h a t e s were u sed . The T4 DNA po lymerase has r e p a i r s y n t h e s i s and 3 ' - e x o n u c l e o l y •a - 3 4 -t i c a c t i v i t y . I t t h e r e f o r e can mod i f y the l a b e l l e d copy s t r a n d f ragment to t e r m i n a t e a t a p o i n t i d e n t i c a l to the 3 ' - e n d of the u n l a b e l l e d t emp l a t e s t r a n d thus d e f i n i n g i t s p o i n t o f c l e a v a g e (see channe l F 1 , F i g . 5 ) . The bands i n c hanne l s F and F 1 ( —• F i g . 5 ) c o - m i g r a t e d , i n d i c a t i n g a f l u s h - e n d e d mode o f c l e a v a g e by Fnu DI i d e n t i c a l to t h a t produced by Hae Hi ( -t> , \ c h a n n e l s H and H 1 r e s p e c t i v e l y ) a t the c e n t r e of the sequence 5 * - G G ^ C C - 3 ' . S i m i l a r e x p e r i m e n t s w i t h Hae I I I f r agment 4 ( a b b r e v i a t e d Z 4 ) as p r i m e r and Hae I I I as datum r e s t r i c t i o n enzyme showed Fnu D i l i to be i d e n t i c a l to Hha I. In t h i s c a s e , the f ragment t r e a t e d w i t h T 4 DNA po l ymera se ( F 1 ) m i g r a t e d a t a f a s t e r r a t e ( 2 n u c l e o t i d e s s h o r t e r than the one i n F channe l ) due to exonuc l eol.y t i c a c i t i v i t y on the 3 ' - e x t e n s i o n a t the c l e a v a g e s i t e . Thus Fnu D i l i i s an i s o s c h i z o m e r o f Hha I both r e c o g -n i z i n g the sequence 3 > I r^GCG-S1 a n ^ c u t t i n g a t t n e p o s i t i o n s i n d i c a t e d by the a r r o w s . A l l e x p e r i m e n t s i n v o l v i n g the Brown and Smith method were r e p e a t e d a t l e a s t once w i t h p r i m e r s f rom d i f f e r e n t r e g i o n s o f the DNA to unambiguous ly prove the n a t u r e of the r e c o g n i t i o n s i t e s . - 35 -F i g . 5. A u t o r a d i o g r a p h of the ge l (12% a c r y l a m i d e i n 7 M urea) f o r the d e t e r m i n a t i o n o f the c l e a vage s i t e o f Fnu DI. In a l l the f i g u r e s i n v o l v i n g DNA s e q u e n c i n g , the p l u s - m i n u s channe l s w i t h the c o r r e s p o n d i n g n u c l e o t i d e s were d e s i g n a t e d as +C, +T, +A, +G, -C , - T , -A and -G r e s p e c t i v e l y . The 0 channe l was the g r a t i c u l e wh ich c o n t a i n s a m i x t u r e of f ragments d i f f e r i n g by a s i n g l e base i n l e n g t h . C leavage by Fnu DI ( i n d i c a t e d by the ar row —• ) was c h a r a c t e r i z e d on channe l s F and F' and compared w i t h the Hae I I I p r o d u c t s ( -^>) on H and H 1 . - 35a -i - 36 -e) Double digestion analysis of Fnu DII Fnu DII d i g e s t e d $X RF DNA gene ra ted a new c l e a v a g e p a t t e r n o f 14 f ragment s whose s i z e s were e s t i m a t e d from s t a n d a r d s o f $X RF DNA d i g e s t e d w i t h Hae I I I and Alu I ( f rom Arthrobaoter luteus) ( F i g . 6 ) . In a d d i t i o n doub le d i g e s t i o n r e s u l t s ( F i g . 6) of Fnu DII and e i t h e r Pst I ( f rom Providenoia stuartii) o r Xho I ( f rom Xanthomonas holicola) were i n a g r e e -ment w i t h computer p r e d i c i t o n s . (McCal1 urn & S m i t h , 1 977) based on the r e v i s e d $X sequence da ta ( Sange r , F., p e r s o n a l communi-c a t i o n ) f o r the r e c o g n i t i o n sequence 5 ' - C G C G - 3 ' . Fnu DII f ragment 7 (259 base p a r i s i n l e n g t h ) was c l e a v e d i n the p re sence of Pst I to two new f ragment s of a p p r o x i m a t e l y 230 and 34 bases l o n g . S i m i l a r l y , the same f ragment was c l e a v e d by xho I to f r agment s 200 and 58 base p a i r s i n l e n g t h . f ) Cleavage mapping of Fnu DII by nearest neighbour analysis To show t h a t r e c o g n i t i o n was u n e q u i v o c a l l y CGCG, the f ragments f rom Fnu DII d i g e s t e d $X DNA were o r d e r e d on the genome by n e a r e s t ne i ghbou r a n a l y s i s as d e s c r i b e d i n the methods and ma.terii.als. L a b e l l e d bands on the a u t o r a d i o g r a p h ( F i g . 7) c o r r e sponded to f ragment s a d j a c e n t to and near the p r i m e r . The c l e a vage map de te rm ined from t h i s p u l s e l a b e l l i n g e xpe r imen t was c o n s i s t e n t w i t h computer p r e d i c t i o n s ( Tab l e 1 ) . - 37 -F i g . 6. A c r y l a m i d e (5%) ge l p a t t e r n s of $X RF DNA d i g e s t e d w i t h the f o l l o w i n g s p e c i f i c e n d o n u c l e a s e s . S l o t -1, Wcm I I I ; 2, Fnu D I I ; 3, Fnu DII and Xho I; 4 , Fnu DII and V&t I; 5, Alu I. fragment sizes (no. of base pairs) I 00 I _ , r O N J r O C J >o C O O O N T * I ^ — — o o o CO cooco K000CO * I I I I I 1 I I I -r [ [ [ [ [ [ r i i m i co c; i IT i r r r r p r r in Ul CO CO CO rO r O Ul NIOO>O zrzi oo^pO>o 00 N T» rO NJrOrO COCO O - ^ U l N CO Ul O ^ V J O O O NJ 00 rO Ul O 00 J K ) CO O - 3 8 -F i g . 7. A u t o r a d i o g r a p h of n e a r e s t ne i g hbo r a n a l y s i s f o r d e t e r m i n i n g ^ the c l e a v a g e map of Fnu DII on $X DNA. The arrows i n d i c a t e the l a b e l l e d f ragment s i m m e d i a t e l y a d j a c e n t to the c o r r e s p o n d i n g p r i m e r f r a g m e n t s . 38a 14 13 12 11 10 9 8 7 6 5 4 3 2 1 primer fragment - 39 -Tab le 1. R e s u l t s of n e a r e s t ne i ghbou r a n a l y s i s ( F i g . 7) compared w i t h computer p r e d i c t i o n s Fragment number 1 S i z e2 N e a r e s t ne i ghbou r f r agment P r e d i c t e d 2 Found ( F i g . 7 ) 1 1050 14 5 3 2 820 4 4 3 718 13 13 4 695 6 6 5 530 7 7 6 496 12 1 , 5 , 6 , 9 , 1 1 , 1 2 3 7 259 10 10 8 170 9 9 9 156 1 1 10 127 3 2 , 3 , 1 2 3 11 114 8 8 12 103 11 11 1 3 79 2 2 14 19 5 -1 Fragments were a c r y l a m i d e g e l s numbered from t h e i r p o s i t i o n on 5% p o l y -2 Ob ta i ned f rom computer p r e d i c t i o n s based on $X DNA sequence data (Sanger et al, 1977a; r e v i s e d , F . Sanger , p e r s o n a l c ommun i ca t i on ) . S i z e i s g i v e n i n number of n u c l e o t i d e s . 3 The appa ren t d i s c r e p a n c y between p r e d i c t e d and found n e a r e s t ne i ghbou r s i n t he se i n s t a n c e s was caused by u n c o n t r o l l e d p u l s e - l a b e l l i n g r e s u l t i n g i n l a b e l l i n g o f more than the immediate n e i g h b o u r . Fragment 14 was not l o c a t e d on the g e l . - 40 -g) Sequencing cleavage s i t e s of Fnu DII F u r t h e r mapping by the Brown and Smith method as d i s c u s s e d i n s e c t i o n d) w i t h Hind I I f r agment 7b (R7b) as p r ime r and Pst I as the datum enzyme, showed t h a t Fnu DII c l e a v e d a t the c e n t r e of the sequence 5 ' -CG" l ' CG-3 ' as i n d i c a t e d by the ar rows ( F i g . 8 ) . A s i m i l a r a c t i v i t y , Tha I has been r e p o r t e d r e c e n t l y (McConnel1 et al, 1 9 78 ) . .- 41 -F i g . 8. A u t o r a d i o g r a p h of Fnu. DII c l e a vage s i t e c h a r a c t e r i z a t i o n by sequence a n a l y s i s ( f o r d e s c r i p t i o n of c h a n n e l s , see F i g . 5 ) . The sequence i n t h i s r e g i o n i s g i v en on the l e f t hand s i d e of the a u t o -r a d i o g r a p h . The c l e a v a g e s i t e of Fnu DII i s i n d i c a t e d by the a r row. - 41a -O I o I I I I I I I I I I I I I " < « i - < u O u O u O « 1 0 t Fnu E I 1 . P u r i f i c a t i o n A s i n g l e e n d o n u c l e o l y t i c a c t i v i t y was i s o l a t e d f rom F. nuoleatum E and was e l u t e d a t 0.39 M KC1 f rom the phospho-c e l l u l o s e column ( F i g . 9) and a t 0.03 M KC1 from D E A E - c e l l u -l o s e column ( F i g . 10) when a l i n e a r g r a d i e n t of 0 -1 .0 M KC1 i n b u f f e r C was u sed . The p u r i f i e d enzyme was c o n c e n t r a t e d a g a i n s t 50% (v/v ) g l y c e r o l c o n t a i n e d i n b u f f e r 0 and s t o r e d a t - 20°C where i t remained s t a b l e f o r ove r 12 months. The p r e p a r a t i o n y i e l d e d about 2,000 u n i t s o f n o n - s p e c i f i c n u c l e a s e f r e e Fnu EI f rom 12 g o f f r o z e n c e l l s . 2. C h a r a c t e r i z a t i o n The enzyme was a c t i v e f o r up to 8 h a t 37°C i n b u f f e r D w i t h maximal a c t i v i t y i n 100 mM NaCI and was i n h i b i t e d by NaCI a t c o n c e n t r a t i o n s g r e a t e r than 300 mM. a) Double digestion analysis Fnu EI d i d not cu t $X RF DNA and o t h e r s u b s t r a t e s were s u b s t i t u t e d . Double d i g e s t i o n on Fnu EI and Mbo I ( f rom Movaxella bovis) on polyoma DNA (a g i f t of Dr. J . C o l t e r ) y i e l d e d no a d d i t i o n a l bands s u g g e s t i n g i s o s c h i z o m e r i c p r o p e r -t i e s ( F i g . 11 , s l o t s 1 -4 ) . However, X DNA d i g e s t e d w i t h Fnu EI showed nove l c l e a v a g e s p e c i f i c i t i e s d i f f e r e n t f rom those found i n Mbo I ( F i g . 11 , s l o t s 8 & 9 ) . F u r t h e r m o r e , doub le d i g e s t i o n w i t h Fnu EI and Alu I on S 1 3 * ' : R F : D N A (a g i f t * a phage w i t h a genome very s i m i l a r to $X174 - 43 -o f Dr. C.A. H u t c h i s o n I I I ) showed c l e a v a g e o f two Alu I f r agments by Fhu-£l not found i n the Mbo l-Aiu I doub le d i g e s t i o n ( F i g . 1 1 , s l o t s 5 - 7 ) . A Fnu EI c l e a v a g e map of S13 RF DNA was e s t a b l i s h e d from f u r t h e r doub le d i g e s t i o n e xpe r imen t s ( F i g . 12, 13; Tab le 2 ) . b) Sequencing the recognition s i t e by 2-D f r a c t i o n a t i o n Mbo I r e c o g n i z e s the sequence 5 ' - ^GATC-3' and c u t s to g i v e f ragments w i t h 5 ' - e x t e n s i o n s as i n d i c a t e d by the a r r ow. I f Fnu EI behaved i n the same manner, then i t s r e c o g n i t i o n s i t e s hou ld be amenable to s e q u e n c i n g . Fragments from Fnu EI d i g e s t e d DNA were l a b e l l e d a t the 5 ' - t e r m i n u s and the base sequence a t the r e c o g n i t i o n s i t e was a n a l y z e d by 2-D f r a c t i o n a t i o n as d e s c r i b e d i n the methods and m a t e r i a l s ( f i g . 14 ) . In o r d e r t o read the sequence on the a u t o r a d i o - r g r aph , the 5 ' - t e r m i n a l base was de te rm ined by t o t a l h y d r o l y s i s w i t h P i f o l l o w e d by paper e l e c t r o p h o r e s i s (see methods and m a t e r i a l s ) . The r e s u l t s showed t h a t most o f the l a b e l was i n c o r p o r a t e d i n t o G and i t was t h e r e f o r e the 5 ' - t e r m i n a l n u c l e o t i d e , i n d i c a t i n g t h a t Fnu EI c l e a v e d i n the same way as Mbo I g e n e r a t i n g a 5 ' - t e r m i n a l G. From the m o b i l i t y s h i f t s on the 2-D f r a c t i o n a t i o n t r a c t , the sequence o b t a i n e d was 5 ' - G A T C - 3 ' . A l l f o u r n u c l o e t i d e s were p r e s e n t beyond the t e t r a n u c l e o t i d e wh ich demons t ra ted t h a t Fnu EI was i ndeed an i s o s c h i z o m e r of Mbo I. - 44 -F i g . 9. Gel e l e c t r o p h o r e s i s a n a l y s i s o f a s say s o f f r a c t i o n s f rom p h o s p h o c e l 1 u l o s e column o f Fnu EI fraction no. - 45 -F i g . 10. Gel e l e c t r o p h o r e s i s a n a l y s i s o f a s say s f r a c t i o n s f rom D E A E - c e l 1 u l o s e column o f Fnu EI - 45a -- 46 -F i g . 11. Compar ison o f Fnu EI w i t h Mbo I d i g e s t i o n p a t t e r n s o f v a r i o u s DNA on 5% a c r y l a m i d e g e l s . Polyoma DNA was used i n s l o t s 1-4, S13 RF DNA i n 5 -7 , and X DNA i n 8 & 9. R e s t r i c t i o n enzymes used were as f o l l o w s : 1, Fnu EI & Mbo I; 2, Mbo I; 3, Fnu E I ; 4, no enzyme added; 5, Mbo I.& Alu I; 6, Fnu EI & Alu I; 7, Alu I; 8, Fnu E I ; 9, Mbo I. The a r rows i n d i c a t e the bands t h a t were c l e a v e d by Fnu EI on S13 RF DNA. - 47 -F i g . 12. Double d i g e s t i o n o f S13 RF DNA w i t h Fnu EI and the v a r i o u s enzymes. S l o t 1, Alu I; 2, Alu I & Fnu E I ; 3, Mbo I I ; 4 , Mbo I I & Fnu E I ; 5, Hha I; 6, Hha I & Fnu E I ; 7, Hae I I I ; 8, Hae I I I & Fnu E I ; 9, Fnu E I ; 10, #ae I I I on $X RF DNA used as s t a n d a r d . The f ragment s i z e s o f the Alu I d i g e s t e d S13 DNA bands were g i v en on the l e f t hand s i d e o f the ge l i n number o f base p a i r s . - 47a -1 2 3 4 5 6 7 8 9 10 - 48 -Tab le 2-. Double d i g e s t i o n o f S I S ' RF DNA w i t h Fnu El and one of the f o l l o w i n g enzymes ( f rom r e s u l t s o f F i g . . 12 ) . Enzyme Fragment c l e a v e d by Fnu E (no. of base p a i r s ) P r oduc t s w i t h Fnu E 2 (no. of base pairs) Ala I 358 215 , 145 276 208 , 56 Taq I 1175 760 , 450 327 220 , 105 Hae II 872 760 , 100 271 = 160, ? fUnd II 1057 = 900 , 210 335 260 , 62 Mbo II 1103 =1000, 101 396 150, 215 Hph I 777 425 , 370 Most of the c l e a v a g e s i t e s on S13 DNA a re i d e n t i c a l to those on §X DNA. The s i z e of the f ragment s quoted here were based $X DNA sequence data ( Sanger , F. et al, 1977; r e v i s e d , Sanger , F. , p e r s o n a l commun ica t ion ) App rox imated from Ala I d i g e s t e d S13 DNA and tfae I I I d i g e s t e d <s>X DNA s t a n d a r d s . - 49 -F i g . 13. C leavage map of Fnu.EI on S13 DNA. The map i s d e r i v e d from r e s t r i c t i o n c l e a v a g e map data of S13 DNA (M. S m i t h , p e r s o n a l c o m m u n i c a t i o n ) . The arrows i n d i c a t e the two l o c a t i o n s of c l e a v a g e by Fnu. EI 1000 2000 i i 3000 4000 5000 • i i Alu I T Taq I ' 8 ' " 4 1 5 1 3 Hae 6 ' 4 1 7 Hind II i M b o II V ' T c 1 8 7 b 7a 4 11b10 11a 1 5 b 5 a ' 8 ' 9 ' ' 6 b ' 5 c ' 6 a 1 5 b ' \ W I ^ 7 1 4 6 4 '11' 3 10 9 5 ' 8 6 WD a Hph I 3 c ' 6 5 3 b ' 4 ' 3 a - 50 -Tab le 3. D e t e r m i n a t i o n of the 5 ' - t e r m i n a l base f o l l o w i n g c l e a v a g e by Fnu E I . The amount of 3 2 P i n c o r p o r a t i o n i n t o the v a r i o u s m o n o n u c l e o t i d e s on X DNA was counted and compared as a pe r cen tage of the t o t a l count s found on the c h r o m a t o g r a p h i c t r a c t . Base % cpm T 14.6 G 61 .4 A 12.7 C 3.0 o r i g i n 8.4 - 51 -F i g . 14. A u t o r a d i o g r a p h of the 2-D f r a c t i o n a t i o n o f o l i g o n u c l e o t i d e s o b t a i n e d from P] p a r t i a l d i g e s t i o n o f A DNA r e s t r i c t i o n f ragment s p roduced by Fnu E I , The f i r s t d imen s i on (1) was e l e c t r o -p h o r e s i s on c e l l u l o s e a c e t a t e a t pH 3 . 5 , and the second d imens i on ( 2 ) , was homochromatography on D E A E - c e l l u l o s e . - 51a -C • ; pGAT pGATC H**M.|HH" r * ^ 1 52 c) Digestion of f l RF DNA B a c t e r i o p h a g e f l dup lex (RF I) DNA (a g i f t o f Dr. G.F. V o v i s ) i s o l a t e d f rom E. c o l i s t r a i n s c o n t a i n i n g d i f f e r e n t DNA methy l a se s were used to r e s o l v e the d i s c r e p a n t b e h a v i o r o f Fnu E I . The E. c o l i dam me thy l a se ( p r e s e n t i n dam+ s t r a i n s ) m e t h y l a t e s f l DNA and r ende r s i t r e s i s t e n t to Mbo I, but s u s c e p t i b l e to i t s i s o s c h i z o m e r Dpn I ( f rom Diploooccus pneumoniae). Phage f l DNA from dam" ho s t i s not m e t h y l a t e d and i s d i g e s t e d by Mbo I but not Dpn I. Fnu EI however, zv. c o m p l e t e l y d i g e s t e d both m e t h y l a t e d and unmethy l a ted f l DNA g e n e r a t i n g a p a t t e r n i d e n t i c a l to Mbo I d i g e s t e d dam" f l DNA and Dpn I d i g e s t e d dam+ f l DNA ( F i g . 1 5 ) . I t s h o u l d be noted t h a t u n l i k e Fnu E I , Dpn I p e r s i s t e n t l y gave p a r t i a l f ragments on dam+ f l RF I DNA due to the p re sence o f dam"I dam ~ h e t e r o d u p l e x s i t e s i n the DNA. Th i s s ugge s ted t h a t dam*I dam h e t e r o d u p l e x c o n s t r u c t e d in vivo would a l s o by c l e a v e d by Fnu E I . - 53 -F i g . 15'. D i g e s t i o n o f f l RF DNA. 1, Mbo I d i g e s t e d dam' f l DNA. 2, Mbo I d i g e s t e d dam+ f l DNA. 3, Dpn I d i g e s t e d dam+ f l DNA. 4 , Fnu EI d i g e s t e d dam' f l DNA. 5, Fnu EI d i g e s t e d dam+ f l DNA. 6, Hae I I I d i g e s t e d $X RF DNA used as a s i z e s t a n d a r d . The f ragment s i z e s were g i v en i n number o f base p a i r s on the r i g h t hand s i de o f the g e l . - 53a -1353 1078 872 603 310 234 - 54 -Fnu CI Fnu CI ( i s o l a t e d from Fusobactevium nucleaturn C) was a l s o an i s o s c h i z o m e r of Mbo I and c l e a v e d o n l y unmethy l a t ed DNA. Fnu CI e l u t e d a t 0.23 M KC1 f rom the p h o s p h o c e l 1 u l o s e column and was not r e t a i n e d by the DEAE-ce l 1 i l l ose column i n b u f f e r C. F r a c t i o n s c o n t a i n i n g enzyme a c t i v i t y were c o n c e n -t r a t e d i n 50% v/v g l y c e r o l i n b u f f e r C. About 500 u n i t s o f a c t i v i t y from 6 g o f f r o z e n c e l l s were o b t a i n e d . L i k e Mbo I» Fnu CI d i g e s t e d DNA gave smeared p a t t e r n s on agarose g e l s making i t d i f f i c u l t to de te rm ine p u r i t y and s a l t e f f e c t s . Double d i g e s t i o n of Fnu CI and Mbo I on po lyoma, \ and T7 DNA gene ra ted no a d d i t i o n a l bands f o l l o w i n g gel e l e c t r o -p h o r e s i s . The 5 ' - t e r m i n a l base gene ra ted from Fnu CI d i g e s t e d A DNA was a n a l y z e d as d e t a i l e d i n the methods and m a t e r i a l s and was found to be G. Fnu CI i s t h e r e f o r e i d e n t i c a l to Mbo I and r e c o g n i z e s the sequence 5 ' - + G A T C - 3 ' c l e a v i n g as shown by the a r r ow . Fnu AI and Fnu A l l With Fusobactevium nucleaturn A, two peaks of a c t i v i t y , Fnu A l and Fnu A l l ,.;were e l u t e d f rom the phos phoce l 1 ul ose column a t a p p r o x i m a t e l y 0.2 M and 0.4 M KC1 r e s p e c t i v e l y . From the D E A E - c e l 1 u l o s e co lumn, Fnu A l was e l u t e d by 200 ml of 0-1 M KC1 T tneac . . g r ad i en t i n b u f f e r C a t 0.05 M KC1 . A c t i v e f r a c t i o n s were d i a l y z e d i n 50% v/v g l y c e r o l i n b u f f e r C - 55 -p r i o r to s t o r a g e a t - 20°C . There i s d i f f i c u l t y i n l o c a t i n g the peak f r a c t i o n s o f Fnu A l l because p a r t i a l c l e a v a g e p a t t e r n s w i t h X DNA p e r s i s t e d . Fnu A l l was not f u r t h e r p u r i f i e d . The p r e p a r a t i o n y i e l d e d about 800-1000 u n i t s of a c t i v i t y from 10 g o f f r o z e n c e l l s . R e s u l t s f rom doub le d i g e s t i o n of <£>X RF DNA showed t h a t Fnu A l was an i s o s c h i z o m e r o f Hinf ( f rom H. influenzae s e r o t y p e f ) wh ich r e c o g n i z e s the sequence 5 ' - G ^ A N T C - 3 1 . Fnu A l c l e a v a g e b e h a v i o r ( c h a n n e l s F and F ' , F i g . 16) was i d e n t i c a l to t h a t o f Hinf ( c hanne l s H and H ' ) as was demons-t r a t e d by the sequenc ing data u s i n g Alu I f r agment 11 ( A l l ) as the p r i m e r and Hae I I I as the datum enzyme. The r e s u l t i l l u s t r a t e d y e t ano the r mode o f r e s t r i c t i o n c l e a v a g e t h a t g ene r a t ed 5 ' - e x t e n s i o n s . In the p re sence p f T4 p o l y m e r a s e , the copy s t r a n d was ex tended to a l o n g e r f ragment by r e p a i r s y n t h e s i s a t the s i t e o f s c i s s i o n as seen i n channe l F 1 ( F i g . 1 6 ) . S i n c e Fnu A l l was not p u r i f i e d , i t was a l s o not c h a r a c t e r i z e d . The enzyme has s i n c e been p u r i f i e d and c h a r a c t e r i z e d as an i s o s c h i z o m e r o f Mbo I (Myer s , P. and R o b e r t s , R . J . , p e r s o n a l c o m m u n i c a t i o n ) . Th i s o b s e r v a t i o n was c o n s i s t e n t w i t h the p a t t e r n s o b t a i n e d from Fnu A l l d i g e s t e d X DNA. - 56 -F i g . 16. A u t o r a d i o g r a p h o f Fnu A l c l e a v a g e c h a r a c t -e r i z a t i o n by sequence a n a l y s i s (see F i g . 5 f o r d e s c r i p t i o n ) . L o c a t i o n of c l e a v a g e s i t e o f Fnu A l was pe r fo rmed i n channe l s F and F 1 , and.Hinf i n H and H 1 . S c i s s i o n on the copy s t r a n d was i n d i c a t e d by the s o l i d a r row and on the t e m p l a t e s t r a n d by the open a r r ow . - 57 -Fnu 48 E n d o n u c l e o l y t i c a c t i v i t y f rom Fusobaoterium nuoleatum 48, was e l u t e d i n f r a c t i o n s 37 to 49 f r o m . t h e B i o g e l A 0.5 m column ( F i g . 1 7 ) . S i n g l e peaks o f a c t i v i t y f o l l o w i n g phos -p h o c e l l u l o s e ( F i g . 1 8 ) . and D E A E - c e l l u l o s e or DEAE-Sephadex ( F i g . 19 ) . were e l u t e d i n 0.05 M, 0.3 M and 0.1 M KC1 i n b u f f e r C r e s p e c t i v e l y . Fnu 48 d i g e s t e d $X DNA gave a un ique p a t t e r n f o l l o w i n g 5% 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 . From doub le d i g e s t i o n e x p e r i m e n t s , t h r e e s i t e s o f c l e a v a g e on $X DNA were l o c a t e d ( f i g . 20, Tab l e 4) and sequenced , i n one case w i t h a E'aeaWl f ragment 4 (Z4) p r i m e r and Hae I I I datum r e s t r i c t i o n enzyme ( F i g . 2 1 ) . From the d a t a , Fnu 48 c l e a v e d a t the c e n t r e o f the sequence S ' - TC^GA-S ' g e n e r a t i n g f l u s h - e n d e d f r a g m e n t s . The same sequence i s r e c o g n i z e d by Taq I ( f rom Thermus aquatious) wh ich cu t s to g i v e 5 1 - e x t e n s i o n s ( 5 ' - T v C G A - 3 1 ) . D i g e s t i o n p a t t e r n s of $X DNA w i t h these two enzymes however were not c ompa rab l e . T h e r e f o r e i n a d d i t i o n to the Taq I i s o s c h i z o m e r i c a c t i v i t y , ano the r r e s t r i c t i o n s p e c i f i c i t y was s u s p e c t e d to be p r e s e n t i n Fnu 48. The sequence r e c o g n i z e d by t h i s a d d i t i o n a l a c t i v i t y remained u n c l e a r . - 58 -F i g . 17. Gel e l e c t r o p h o r e s i s a n a l y s i s o f a s say s f r a c t i o n s from B i o g e l column o f Fnu 48 - 58a -- 59 -F i g . 18. Assays o f f r a c t i o n s from p h o s p h o c e l 1 u l o s e column o f Fnu 48 a n a l y z e d by gel e l e c t r o p h o r e s i s . f r a c t i o n no. 11 17 23 29 47 53 59 65 - 60 -F i g . 19 Assay o f f r a c t i o n s f rom DEAE-Sephadex column o f Fnu 48 by ge l e l e c t r o p h o r e s i s a n a l y s i s . f r a c t i o n no. 15 17 19 21 23 25 27 29 31 33 - 61 -F i g . 20. Double d i g e s t i o n o f $X RF DNA w i t h Fnu 48 and v a r i o u s enzymes. S l o t 1,LRae I I I ; 2, Hae I I I & Fnu 48; 3, F i t I & Fnu:.48; 4, Fnu 48; 5, Xho I & Fnu 48. The ge l p a t t e r n s o f Fnu 48 d i g e s t e d <£>X RF DNA tend to va ry a t the p o s i t i o n s i n d i c a t e d by the open arrows due to p e r s i s t e n t p a r t i a l c l e a v a g e o f one o f the bands. The s o l i d ar row showed the band c l e a v e d by Pst I. - 61a -- 62 -Tab l e 4. Double d i g e s t i o n of $X RF DNA w i t h Fnu 48 and one of the f o l l o w i n g enzyme (base on r e s u l t s from F i g . 2 0 ) . Enzyme Fragment c l e a v e d by Fnu 48 , (no. of base p a i r s ) P r oduc t s w i t h Fnu 48 2 (no. of base pairs) Haz I I I 194 234 170 o r 145 145, 85 Enzyme • !. Fnu 48 f ragment c l e a v e d ^ (no. of base p a i r s ) P r oduc t s (no. of base pairs) ?i>t I 87 50 Kho I not c l e a v e d no a d d i t i o n a l bands T See f o o t n o t e s of t a b l e 2. - 63 -F i g . 21. A u t o r a d i o g r a p h o f the ge l f o r d e t e r m i n a t i o n o f c l e a v a g e s i t e o f Fnu 48 by Brown and Smith method ( f o r d e s c r i p t i o n , see F i g . 5 ) . Channel s H c o n t a i n e d a l a b e l l e d f ragment (open ar row) from Hha I c l e a v a g e and s e r v e d as a marker f o r l o c a t i n g the r e g i o n on the $X DNA t h a t was sequenced. The sequence o b t a i n e d was g i v e n on the l e f t hand s i d e o f the ge l and the p o i n t o f c l e a vage by Fnu 48 was i n d i c a t e d by the s o l i d a r r ow . - 63a -O +C +T +A +G -C -T -A -G F F' H - 64 -Mi s c e l l a n e o u s Be s i de the Fusobactevium, o t h e r b a c t e r i a have a l s o been examined f o r the p re sence o f r e s t r i c t i o n enzymes. Th i s i n c l u d e s two f a c u l t a t i v e anae robe s , Streptococcus sanguis and Streptococcus m i t i s , and one s t r i c t anae robe , Bacteroides melaninogenicus s u b s p e c i e s melaninogenicus. No r e s t r i c t i o n a c t i v i t y was d e t e c t e d i n any of these b a c t e r i a a l t h o u g h a p o t e n t n u c l e o l y t i c a c t i v i t y was found i n the B. melaninogenicus ( F i g . 2 2 ) . - 65 -F i g . 22. Assay o f f r a c t i o n s from B i o g e l A 0.5 m column o f the h igh speed s u p e r n a t a n t o f Baoteroides meldninogenieus c e l l e x t r a c t s , b y ge l e l e c t r o p h o r e s i s a n a l y s i s . - 65a -- 66 -DISCUSSION Of the a n a e r o b i c b a c t e r i a t h a t have been examined f o r the p re sence of r e s t r i c t i o n enzymes, s p e c i f i c e n d o n u c l e o l y t i c a c t i v i t y i s d e t e c t e d o n l y i n the Fusobaoterium genus. F a i l u r e to d e t e c t r e s t r i c t i o n a c t i v i t y does not i m p l y t h a t such enzymes are absent i n the o r gan i sm as the a b i l i t y to d e t e c t endonuc l ea se s i s dependent on many f a c t o r s . R e s t r i c t -i o n enzymes may not be r e l e a s e d f rom organ i sms t h a t a re r e s i s t e n t to m e c h a n i c a l b r eakage ; o r i f r e l e a s e d , they may be i n h i b i t e d by the s a l t c o n c e n t r a t i o n s (200 mM) i n the a s says o f B i o g e l column f r a c t i o n s , by o t h e r i n h i b i t o r s , o r c o n v e r s e l y , the enzymes may have c e r t a i n c o f a c t o r r e q u i r e m e n t s not s u p p l i e d i n the r e a c t i o n m i x t u r e . F u r t h e r m o r e , the p re sence of r e s t r i c t i o n a c t i v i t y may be o v e r l o o k e d i n cases where the enzymes f a i l to c l e a v e the \ DNA, f a i l to produce DNA f ragment s t h a t are r e s o l v a b l e on the 1.4% agarose ge l , a re ob scu red by e x c e s s i v e amounts o f n o n - s p e c i f i c n u c l e a s e s , ot a re p r e s e n t i n u n d e t e c t a b l y low amounts. T;o overcome problems of h igh s a l t and n o n - s p e c i f i c n u c l e a s e s , s m a l l e r a l i q u o t s ( e . g . 2 y l i n s t e a d o f 5 y l ) can be used i n the a s say . Where A DNA i s s u spec ted to a poor s u b s t r a t e , o t h e r DNAs can be s u b s t i t u t e d . Th i s s tudy i s l i m i t e d to enzymes t h a t are d e t e c t e d under the as say c o n d i t i o n s g i v e n i n the methods and m a t e r i a l s f o l l o w i n g the f i r s t c h r o m a t o g r a p h i c s t e p . In cases - 67 -where no a c t i v i t y i s d e t e c t e d , the e xpe r imen t s s h o u l d be r e p e a t e d u s i n g d i f f e r e n t a s say c o n d i t i o n s and p u r i f i c a t i o n methods. The s e a r c h f o r "new" r e s t r i c t i o n endnocnu l cea se s i n v o l v e s enzymes w i t h nove l sequence r e c o g n i t i o n as w e l l as "new" s ou rce s of a c t i v i t i e s t h a t s u b s t i t u t e i s o s c h i z o m e r s found i n poor p r o d u c i n g o r p a t h o g e n i c s t r a i n s . To both these ends , the Fusobactevium nuoleatum have been r e w a r d i n g l y p r o d u c t i v e . From f i v e s t r a i n s o f t h i s anaerobe i s o l a t e d f rom the o r a l c a v i t y o f v a r i o u s i n d i v i d u a l s , seven enzymes w i t h d i f f e r e n t s p e c i f i c i t i e s are r e p o r t e d . Th i s r e s u l t sugges t s a ve r y h i gh p r o b a b i l i t y o f f i n d i n g d i f f e r e n t a c t i v i t i e s i n s t r a i n s " , i s o l a t e d from d i f f e r e n t peop le and p r e d i c t s the d i s c o v e r y o f more enzymes i n the s p e c i e s o r even i n the genus. Fusobactevium nuoleatum i s n o n - p a t h o g e n i c , grows to h i gh d e n s i t i e s (3g o f c e l l s per l i t r e o f c u l t u r e ) i n l a r g e volumes w i t h o u t a e r a t i o n , c o n t a i n s l i t t l e n o n - s p e c i f i c n u c l e a s e s t h a t i n t e r f e r e s w i t h the p u r i f i c a t i o n o f r e s t r i c t — i o n enzymes i n most i n s t a n c e s , and i t s n ded l e - s haped morphology r ende r s these b a c t e r i a s u s c e p t i b l e to d i s i n t e g r a -t i o n w i t h m i l d s o n i c a t i o n . A l l t he se f a c t o r s add to the advantages o f the Fusobactevium as a good sou rce f o r . . . r e s t r i c t i o n enzymes. A l i s t o f the Fnu enzymes and t h e i r c l e a v a g e on $X DNA i s g i v en i n Tab le 5 and F i g . 23. Tab le 5. The s i z e s of f r agment s generated from c l eavage of <l>X174am3 DNA by Fnu enzymes Fnu A I Fnu DI Fnu DII Fnu D i l i Fnu 481 Fragment Fragment Fragment Fra gmen t Fragment number S i z e1 number S i z e1 n umber S i z e1 number S i z e1 number S i z e1 1 726 1 1 353 1 105 0 1 1553 1 2914 2 713 2 1078 2 870 2 640 2 11 75 2 3 553 3 872 3 718 3 610 3 404 4 506 4 603 4 695 4 532 4 327 5a 427 5 310 5 530 5 305 5 231 5b 413 6a 271 6 496 6 300 6 141 6 311 6b 281 7 259 7 269 7 87 7 249 7 234 8 170 8a 201 8 54 8 206 8 194 9 156 8b 192 9 33 9 151 9 118 10 127 9a 1 45 10 20 10 140 1 0 72 11 114 9b 143 11 118 12 103 10 123 12 100 13 79 11 101 13 82 14 19 12 93 14a,b 66 13 84 1 5 48 14 54 1 6a ,b 42,4-0 15 35 17 24 S i z e was g i v e n i n number o f base p a i r s and was based on computer p r e d i c t i o n s u s i n g cj>X174am3 sequenc ing da ta (Sanger et al 1 977a; r e v i s e d , F. Sanger , p e r s o n a l commun ica t i on ) Th i s f r agment was c l e a v e d by an unknown a c t i v i t y i n Fnu 4811 to two f ragments a p p r o x i m a t e l y 600 and 575 bases l ong - 70 -F i g . 23. C leavage map o f Fnu enzymes on $X174am3 DNA. 1000 I 2000 i 3000 4000 i 5000 Fnu AI GANTC J_i l 13 17 16 16 14 I 9 , W 1 14 12 I I 2 |11| 7 i 5 b i 8 i 4 i i i 1011 5 c | 3 ( 5 o [ 6 Fnu D I GGCC 7 ' 5 ' 8 i 1 6 b | 6 o | 9 ,iq 3 Fnu DII CGCG 1 | 9 | 8 ,10,11, 6 , 4 2 ,12. 3 Fnu DIN GCGC 8 b , 4 | ^ , 1 0 , 7 | 5 , 9 a , 8 a , 6 , 3 , 2 , 9 b I I I I l ' w l I  j 8 a j J 1 L Fnu 48 TCGA 8 9 10 7 1 I'" A 5 , 3 18 22 25 16 23 25 Fnu 4H GcVlGC 7 • 3 5 ^ • ' •'• 8 ' 1 2 ' 4 , 6 , 6 ,ii,',l4tir 1 , 24 20 19 2 I 9l'< 5 I 1 0 ! 1 5 ! ' . ' ! 1 0 - 71 -Fnu A l , C I , DI and D i l i a re i d e n t i c a l i n r e c o g n i t i o n and c l e a v a g e to Hinf, Mbo I, Hae I I I and ff/za I r e s p e c t i v e l y . Fnu 48 t h a t c l e a v e s Taq I f r agment 2 once (on $X DNA) appear s to r e c o g n i z e a sequence a t a p p r o x i m a t e l y 4783 bases from the Pst I c u t on $X DNA j u d g i n g from the band ing p a t t e r n s on 5% 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 ( Leung , D. , p e r s o n a l communica-t i o n ) . One l i k e l y c a n d i d a t e t h a t i s c o n s i s t e n t w i t h t h i s r e s u l t i s the h e x a n u c l e o t i d e 5 1 -GTGCAC-3 ' , a nove l sequence t h a t o c c u r s once on the $X genome between 4778 to 4784 bases from the Pst I c u t . There has been d i f f i c u l t y i n o b t a i n i n g s equenc ing da ta i n t h i s r e g i o n o f t h e g e n o m e and the e x a c t r e c o g n i t i o n sequence o f the a d d i t i o n a l a c t i v i t y i n Fnu 48 reamins o b s c u r e . Novel s p e c i f i c i t i e s are found i n Fnu DII ( 5 1 - C G t c G - 3 ' ) and i n Fnu 4H (5 1 -GC^NGC-3 1 ) ( Leung , D. , p e r s o n a l communicat-•> i o n ) . The enzyme Fnu DII i s e xpec t ed to g i ve l i m i t e d c l e a v a g e on e u k a r y o t i c DNA such as the SV40 genome i n wh ich the sequence CG o c c u r s ve ry i n f r e q u e n t l y ( F i e r s et al, 1 978 ) . One enzyme w i t h un ique s p e c i f i c i t y i s the Fnu E I , an i s o -s ch i zomer of Mbo I but has the c h a r a c t e r i s t i c o f r e c o g n i z i n g both m o d i f i e d and u n m o d i f i e d DNA c o n t a i n i n g the sequence 5 ' - G A T C - 3 ' . Dpn I r e c o g n i z e s the same sequence o n l y when the aden ine i s m e t h y l a t e d a t the N-6 p o s i t i o n . Fnu EI w i t h i t s d i v e r s e s u b s t r a t e range has been e x p l o i t e d i n s e v e r a l l a b o r a -t o r i e s f o r d i g e s t i n g m o d i f i e d r e g i o n s of the DNA ( O t s u k a , A. - 72 -and A b e l s o n , J . , p e r s o n a l commun ica t i on ) as w e l l as SPOT DNA wh ich c o n t a i n s h y d r o x y m e t h y l u r a c i 1 ( B r enna , S., p e r s o n a l c o m m u n i c a t i o n ) . . SPOT DNA i s a l s o d i g e s t e d by Eoo R I , Hind I I I and Hpa I I , a l l o f wh ich c l e a v e s u n m o d i f i e d DNA as w e l l . The a b i l i t y to r e c o g n i z e both m o d i f i e d (N-6 me thy l aden i ne or 5 ' -h y d r o x y m e t h y l u r a c i 1 ) and u n m o d i f i e d DNA l e ad s one to s p e c u l a t e an i n t e r a c t i o n o f these enzymes w i t h the minor groove of the DNA h e l i x . Most bases are p r o t e c t e d w i t h i n the h e l i c a l s t r u c t u r e of the DNA excep t f o r the 6 and 8 p o s i t i o n s of .. p u r i n e s and the 4 and 5 p o s i t i o n s of the p y r i m i d i n e s wh ich are exposed i n the major groove (We l l s et al, 1977 ) . Thus i f the r e c o g n i t i o n were to o c c u r a t the minor g r oo ve , a l t e r a t i o n s i n these p o s i t i o n s o f the DNA s hou l d not a f f e c t the c l e a vage b e h a v i o r o f the enzymes. C o n v e r s e l y , enzymes t h a t do not c l e a v e DNA m o d i f i e d i n these p o s i t i o n s may r e q u i r e i n t e r a c -t i o n w i t h the major groove f o r s c i s s i o n . E l u c i d a t i o n of the mechanisms i n v o l v e d i n the r e c o g n i t i o n and c l e a v a g e of DNA by r e s t r i c t i o n enzymes i n e v i t a b l y r e q u i r e s i n f o r m a t i o n on the k i n e t i c s o f enzyme-DNA i n t e r a c t -i on as w e l l as knowledge on the n a t u r e o f the p r o t e i n m o l e c u l e i n q u e s t i o n . With the e x c e p t i o n of Eco R I , no o t h e r r e s t r i c t -i on enzymes have been p u r i f i e d to homogene i ty . Eoo RI has a m o l e c u l a r we i gh t o f 59,000 d a l t o n s , w i t h s u b u n i t s o f 29,500 d a l t o n s (Boyer et al, 1974) arid r e c o g n i z e s the h e x a n u c l e o t i d e - 73 -5 ' -GAATTC-3 ' , a l t h o u g h bases o u t s i d e t h i s sequence do \ . i n f l u e n c e the r a t e of c l e a v a g e (Thomas & D a v i s , 1975) . S t e r i c h i n d r a n c e i n the major groove of the DNA h e l i x i s .. e xpec t ed to be g r e a t e r when the sequence i s found a d j a c e n t to A-T base p a i r s than G-C base p a i r s and vice versa i n the minor groove ( J o v i n , 1976) . The hexanuc l e o t i de s p e c i f i c i t y , i s r e l a x e d under a l t e r e d pH and i o n i c c o n d i t i o n s ( P o l i s k y et al, 1975) . In g e n e r a l , most r e s t r i c t i o n enzymes show a broad pH and M g + + opt imum, are i n h i b i t e d by h igh c o n c e n t r a t i o n s o f N a C l , and are ve r y s t a b l e . T h i s l a s t p r o p e r t y demons t r a te s t h a t the enzymes are not o n l y i n t r i n s i c a l l y s t a b l e but t h a t the p r e p a r a t i o n s are a l s o r e l a t i v e l y f r e e o f p r o t e a s e s . The a c t u a l mechan i sms, t h a t are o p e r a t i v e i n the enzymes remain s p e c u l a t i v e e x cep t t h a t t h e i r s p e c i f i c i t i e s are c o n f e r r e d by n u c l e o t i d e sequences 4 to 6 bases l ong and not by the t o p o l o g y of the DNA m o l e c u l e . Work on the i n t e r a c t i o n of r e s t r i c t i o n enzymes and DNA w i l l u ndoub ted l y shed l i g h t on the r e g u l a t i o n of genome e x p r e s s i o n . R e s t r i c t i o n endonuc l ea se s are not o n l y i n t r i g u i n g m o l e c u l e s but a re a l s o i n v a l u a b l e t o o l s t h a t a re i n s t r u m e n t a l i n the advances o f g e n e t i c e n g i n e e r i n g , s t u d y i n g gene s t r u c t -ure and f u n c t i o n and DNA sequence a n a l y s i s . As t o o l s , t he se enzymes p l a y a dual r o l e i n d i g e s t i n g DNA f o r ? a n a l y t i c a l - 74 -purposes as w e l l as m e d i a t i n g DNA r e c o m b i n a t i o n i n g e n e t i c e n g i n e e r i n g . A l t hough in v i t r o use o f these enzymes have been f u l l y e x p l o i t e d , l i t t l e i s known of t h e i r r o l e in vivo. I t i s g e n e r a l l y a c c e p t e d t h a t the enzymes f u n c t i o n to p r o t e c t the organ i sm a g a i n s t f o r e i g n DNA th rough r e s t r i c t i o n and m o d i f i c a t i o n p r o c e s s e s . Yet no r e s t r i c t i o n of f o r e i g n DNA can be d e t e c t e d i n some b a c t e r i a l s p e c i e s t h a t c o n t a i n the enzymes. What then i s the p r i m a r y f u n c t i o n of t he se enzymes i n these i n s t a n c e s ? . C a n . i t " b e . DNA r e c o m b i n a t i o n ? P r e l i m i n -a ry e xpe r imen t s have shown t h a t the i n s e r t i o n of the Eoo RI gene i n t o an o rgan i sm promotes s i t e - s p e c i f i c r e c o m b i n a t i o n event s in vivo (Chang & Cohen, 1977 ) . A l t h o u g h the f r e q u e n c y o f such even t s are e x t r e m e l y l ow , t h e i r impo r t ance cannot be u n d e r e s t i m a t e d and tremendous i m p l i c a t i o n s of in vivo recomb-i n a t i o n on b i o l o g i c a l e v o l u t i o n a re a l r e a d y e v i d e n t . C l e a r l y t h e r e are many f a c e t s of r e s t r i c t i o n enzymes t h a t a w a i t e x p l o r a t i o n . In our l a b o r a t o r y , the goa l i s towards the d i s c o v e r y of b e t t e r sou rce s of enzymes as w e l l as enzymes w i t h nove l s p e c i f i c i t i e s . Th i s t a sk w i l l be g r e a t l y f a c i l i t a t e d by the new p u r i f i c a t i o n p rocedu re f o r r e s t r i c t i o n enzymes t h a t uses a s i n g l e column of C i b a c r o n b l ue F3GA Sepharose ( Bak s i & R u s h i z k y , 1978 ) . The p r o c e d u r e has been t e s t e d out w i t h a p r e p a r a t i o n o f Fnu 48 and a p u r i f i c a t i o n comparab le to the c o n v e n t i o n a l 3-column method i s a c c o m p l i s h e d (Leung, D., p e r s o n a l c o m m u n i c a t i o n ) . - 75 -Once p u r i f i e d , sequence r e c o g n i t i o n and c l e a v a g e o f the enzymes t h a t d i g e s t $X DNA can be r e a d i l y c h a r a c t e r i z e d by the Brown and Smith method f o l l o w i n g the l o c a t i o n o f one o r two of these s i t e s by doub le d i g e s t i o n e x p e r i m e n t s . I n c r e a s -i n g sequencing data t h a t a re becoming a v a i l a b l e on o t h e r v i r a l genomes sugges t t h a t such c h a r a c t e r i z a t i o n methods are not o n l y l i m i t e d to enzymes t h a t c l e a v e $X DNA but a re p o t e n t i a l l y a p p l i c a b l e to c h a r a c t e r i z a t i o n of a l l C l a s s II r e s t r i c t i o n e n d o n u c l e a s e s . Compar ison o f computer p r e d i c t e d f r a g m e n t a t i o n from sequenc i ng data w i t h the p a t t e r n s o b t a i n e d i n gel e l e c t r o p h o r e s i s he lp s to c o n f i r m the r e c o g n i t i o n sequence. With i m p r o v i n g methods, the i s o l a t i o n and c h a r a c t e r i z a -t i o n o f r e s t r i c t i o n enzymes w i l l be g r e a t l y f a c i l i t a t e d . 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