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Studies on Acetabularia chloroplast DNA Muir, Bernice L. 1974

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STUDIES ON  ACETABULABIA CHLOROPLAST DNA  by BERNICE L. MUIR B.Sc., U n i v e r s i t y o f B r i t i s h Columbia, 1970  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n the Department of Botany  We accept t h i s  t h e s i s as conforming t o the  required standard  THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1974  In p r e s e n t i n g an the  advanced degree at Library  I further for  this thesis  shall  by  his  of  this thesis  of  p u r p o s e s may  be  g r a n t e d by  shall  8.  Botany  1974  the  Columbia  not  the  requirements  Columbia,  for reference  Head o f my  be  I agree and  copying of  It i s understood that  for f i n a n c i a l gain  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a  April  British  for extensive  permission.  Department o f  Date  University  permission  representatives.  written  f u l f i l m e n t of  make i t f r e e l y a v a i l a b l e  agree t h a t  scholarly  the  in partial  that  study.  this  thesis  Department  copying or  for  or  publication  allowed without  my  ii  ABSTRACT  The p h y s i c a l p r o p e r t i e s and r e n a t u r a t i o n k i n e t i c s o f DNA e x t r a c t e d from i s o l a t e d c h l o r o p l a s t s o f  Acetabulavia  meditewanea  has been s t u d i e d .  3 I t has a buoyant d e n s i t y o f 1.702 g/cm , which corresponds p o s i t i o n o f 42.8% G+C. has  When melted  i n SSC,  chloroplast  a Tm o f 86.7°, c o r r e s p o n d i n g t o a base composition o f 43% G+C.  c l o s e agreement o f the base compositions s i t y and the m e l t i n g temperature in  Acetabulavia  t o a base com-  Acetdbularia  meditewanea  In 0.1 x SSC,  c a l c u l a t e d from the buoyant den-  DNA.  c h l o r o p l a s t DNA melts w i t h a T  and the m e l t i n g t r a n s i t i o n i s v e r y broad. s i t i o n suggests geneity.  The  i n d i c a t e s the absence o f unusual bases  chloroplast  Acetabulavia  DNA  m  o f 70.7°,  The b r e a d t h o f t h e m e l t i n g  tran-  t h a t t h i s DNA has a h i g h degree o f i n t r a m o l e c u l a r h e t e r o -  A d i f f e r e n t i a l p l o t o f the thermal t r a n s i t i o n o f  A.  meditewanea  c h l o r o p l a s t DNA supports t h i s c o n c l u s i o n . The buoyant d e n s i t i e s o f DNA from b a c t e r i a l contaminants  Acetabulavia DNA.  found i n  c u l t u r e s d i f f e r e d from the buoyant d e n s i t y o f the c h l o r o p l a s t  I n any case, the amount o f b a c t e r i a l c o n t a m i n a t i o n was too low to  account  f o r any o f the r e s u l t s  obtained.  9 R e n a t u r a t i o n experiments d a l t o n s from  Acetabulavia  i n d i c a t e a k i n e t i c complexity o f 1.1 x 10  meditewanea  c h l o r o p l a s t DNA.  As a r e s u l t o f un-  c e r t a i n t i e s i n the v a l u e s o f a l k a l i n e s e d i m e n t a t i o n c o e f f i c i e n t s , t h i s  cal-  c u l a t e d k i n e t i c complexity may be too low. The p o s s i b l e g e n e t i c i n f o r m a t i o n c o n t a i n e d i n the c h l o r o p l a s t DNA o f  Acetabulavia  meditewanea  i s discussed.  iii  TABLE OF CONTENTS  Page INTRODUCTION  1  MATERIALS AND METHODS  7  Materials  7  Treatment o f c y s t s p r i o r t o g e r m i n a t i o n  7  Cultivation Isolation  and treatment o f a l g a e  ...  of c h l o r o p l a s t s  10  P r e p a r a t i o n o f c h l o r o p l a s t DNA Monitoring f o r b a c t e r i a l Isolation  10  ••  12  contamination....  o f DNA from b a c t e r i a l  14  contaminants..  Cesium c h l o r i d e d e n s i t y g r a d i e n t s  15  •  16  E l e c t r o n microscopy  16  P r e p a r a t i o n o f E. aoli  DNA  16  P r e p a r a t i o n o f b a c t e r i o p h a g e T^ DNA  .  17  Thermal d e n a t u r a t i o n p r o f i l e s and d e t e r m i n a t i o n o f T .. m R e n a t u r a t i o n o f DNA •  18  Determination of a l k a l i n e  20  r  RESULTS  sedimentation c o e f f i c i e n t s . . . ...  22  Attempts t o o b t a i n a x e n i c c u l t u r e s o f Bacterial  Aeetabulavia  contamination i n c h l o r o p l a s t p r e p a r a t i o n s . . . .  E l e c t r o n microscopy  18  •.  22 23 25  Buoyant d e n s i t y and T o f Aeetabulavia c h l o r o p l a s t DNA. R e n a t u r a t i o n and k i n e t i c c o m p l e x i t y o f Aeetabulavia c h l o r o p l a s t DNA  27  Sedimentation c o e f f i c i e n t s . . . . . . . . .  38  m  DISCUSSION  32  41  iv  LIST OF  FIGURES Page  F i g u r e 1.  Figure  2.  E l e c t r o n micrograph o f t h i n s e c t i o n s of Aeetabulavia meditevvanea c h l o r o p l a s t s C s C l buoyant d e n s i t y  Aeetabulavia Figure  3.  F i g u r e 4.  Figure  5.  F i g u r e 6.  c e n t r i f u g a t i o n of  meditevvanea c h l o r o p l a s t DNA  M e l t i n g p r o f i l e s of Aeetabulavia c h l o r o p l a s t DNA and E. coli DNA D i f f e r e n t i a l p l o t of the  28  meditevvanea  thermal t r a n s i t i o n c h l o r o p l a s t DNA  Aeetabulavia meditevvanea and E. coli DNA  Renaturation rate p l o t s for Aeetabulavia meditevvanea c h l o r o p l a s t DNA.... M e l t i n g p r o f i l e s of r e n a t u r e d Aeetabulavia meditevvanea c h l o r o p l a s t DNA and r e n a t u r e d  E. eoli DNA  26  30 of  31 33  35  V  LIST OF TABLES  Page Table I.  Table I I .  Table I I I .  Table IV.  T a b l e V.  B a c t e r i a l contaminants preparations.  of  K i n e t i c c o m p l e x i t i e s o f DNA sources  chloroplast 24 from d i f f e r e n t 36  A l k a l i n e sedimentation c o e f f i c i e n t s of DNA samples  sheared  V a r i a t i o n i n k i n e t i c complexity w i t h d i f f e r e n t v a l u e s of SPH13 20 ,w K i n e t i c c o m p l e x i t i e s of c h l o r o p l a s t v a r i o u s sources  39  40  DNAs from 44  vi  ACKNOWLEDGMENTS  I wish t o thank Dr. B.R. Green f o r many h e l p f u l  discussions  throughout the course of t h i s work, and f o r guidance i n p r e p a r i n g thesis.  this  T e c h n i c a l a s s i s t a n c e from Mrs. V a l e r i e Macdonald, Mrs. Margaret  Ward and Mrs. L i v i a Beck was g r e a t l y a p p r e c i a t e d .  I a l s o wish t o thank  the Canada Department o f A g r i c u l t u r e , Dr. S.H. Zbarsky o f t h e B i o c h e m i s t r y Department, D r . J . Levy and Dr. J . J . Stock o f t h e M i c r o b i o l o g y  Department,  Dr. H. K a s i n s k y o f t h e Zoology Department and Dr. A. Bree o f t h e Chemistry Department, U.B.C, f o r g e n e r o u s l y a l l o w i n g me t h e use o f equipment. L a s t , b u t n o t l e a s t , I w i s h to acknowledge t h e 51,570 t h a t gave t h e i r l i v e s i n t h e cause o f s c i e n c e . s e a r c h would n o t have been p o s s i b l e .  Acetabulavia  cells  Without them, t h i s r e -  INTRODUCTION  The p u r p o s e o f t h i s r e s e a r c h w a s t o d e t e r m i n e  the complexity o f  Aeetabulavia  meditevvanea  c h l o r o p l a s t DNA f r o m i t s r e n a t u r a t i o n k i n e t i c s .  Aeetabulavia  meditevvanea  has been used e x t e n s i v e l y t o study  nucleo-  cytoplasmic i n t e r a c t i o n s and, thus, i t i s o f i n t e r e s t t o determine  some-  t h i n g a b o u t t h e g e n e t i c p o t e n t i a l o f t h e c h l o r o p l a s t DNA. A l s o , w i t h Aeetabulavia  i t i s p o s s i b l e t o o b t a i n c h l o r o p l a s t DNA w h i c h i s c o m p l e t e l y  free from contamination meditevvanea  b y n u c l e a r DNA.  was chosen f o r t h i s  F o r these reasons,  Aeetabulavia  research.  Renaturation k i n e t i c s are useful f o rstudying the complexity and, t h e r e f o r e , t h e g e n e t i c p o t e n t i a l o f DNA f r o m a p a r t i c u l a r s o u r c e . be denatured  b y heat, a l k a l i o rvarious chemicals,  separation o f t h e complementary strands. temperature,  DNA may  r e s u l t i n g i n complete  Under appropriate c o n d i t i o n s o f  i o n i c s t r e n g t h and pH, t h e separated  come b a c k t o g e t h e r i n r e g i s t e r , i . e . r e n a t u r e .  complementary  strands  Theconditions f o r renatura-  t i o n and t h e k i n e t i c s o f t h e r e a c t i o n have been s t u d i e d e x t e n s i v e l y b y various researchers  (12,13,14,40,45,65,68,74) a n d m a y b e s u m m a r i z e d a s  follows: 1)  The r e n a t u r a t i o n r e a c t i o n i s a second order  2)  The second-order  rate constant  proportional t othe complexity  process  f o rrenaturation i s inversely o f t h e DNA, w h e r e  complexity  i s d e f i n e d a s t h e t o t a l number o f DNA b a s e p a i r s i n ing 3)  non-repeat-  sequences.  Theoptimum temperature  f o r r e n a t u r a t i o n i s 20° t o 30°C b e l o w  the melting temperature  ( T ) o f t h e DNA. m  2  4)  The DNA r e n a t u r a t i o n r a t e i s dependent on i o n i c  strength  of t h e s o l v e n t f o r an e l e c t r o l y t e such as N a C l . 5)  Decreasing  the m o l e c u l a r  weight o f a g i v e n DNA r e s u l t s i n  a decrease i n the r a t e o f r e n a t u r a t i o n o f the DNA. 6)  The r a t e o f r e n a t u r a t i o n i n c r e a s e s s l i g h t l y w i t h GC  7)  increasing  content.  The r a t e o f r e n a t u r a t i o n o f DNA depends on s o l v e n t  viscosity.  + 8)  Within  the pH range 5 t o 9 i n 0.4 M Na  the r a t e o f r e n a t u r a -  t i o n i s e s s e n t i a l l y independent o f pH. 9)  10)  C o r r e c t base sequence matching can occur  to a greater  when the DNA i s sheared t o low m o l e c u l a r  weight.  extent  The thermal s t a b i l i t y o f t h e DNA i s decreased about one degree f o r 1.5 p e r cent m i s p a i r e d bases i n t h e r e n a t u r e d  DNA.  Wetmur and Davidson (74) have d e r i v e d t h e f o l l o w i n g e q u a t i o n mining the k i n e t i c complexity  ND n  =  o f DNA.  331N =  5 . 5 . x . l 0 (ZUS ?,wS ) 8  k  where N^ i s the complexity r a t e constant  f o r deter-  1 3  1 , 2 5  2  o f the DNA i n d a l t o n s ,  f o r r e n a t u r a t i o n o f the DNA: and  i s t h e second  S?|P"^  order  i s the a l k a l i n e  sedi-  20 ,w  mentation c o e f f i c i e n t o f the DNA. t i o n r a t e constant  This equation  i s v a l i d when the r e n a t u r a -  i s determined a t [ N a J = 1.0 mole 1 ^ i n aqueous s o l u t i o n +  at a temperature near (T -25)°C. m  Acetabulavia Since  has been an important r e s e a r c h organism f o r two r e a s o n s .  t h e 1930's, t h i s marine a l g a has been used e x t e n s i v e l y t o study  3 nucleo-cytoplasmic i n t e r a c t i o n s . the f i r s t p l a n t i n which i t was of  DNA  of  p o s s i b l e t o demonstrate  Aeetabulavia  med'itevvanea,  a s i n g l e c e l l may  3 to 5 cm i n a p e r i o d of 3 months.  life  Aeetabulavia  became  the e x i s t e n c e  i n the c h l o r o p l a s t s .  With of  More r e c e n t l y ,  achieve a length  During t h i s v e g e t a t i v e p a r t o f the  c y c l e a s i n g l e , l a r g e n u c l e u s i s l o c a t e d i n the r h i z o i d a t the base  the c e l l .  Growth o f the c e l l c u l m i n a t e s i n the f o r m a t i o n o f a r e p r o -  d u c t i v e cap, which takes about t h i s time may  be 0.3  to 0.5  diameter o f up to one cm.  mm,  one month.  The diameter o f the s t a l k at  w h i l e the f u l l grown cap may  When the cap i s f u l l y  n u c l e u s d i v i d e s to form s e v e r a l thousand  attain a  grown, the l a r g e  primary  s m a l l , secondary n u c l e i .  By  means o f c y t o p l a s m i c streaming, the secondary n u c l e i are t r a n s p o r t e d from the r h i z o i d t o the cap, where c y s t s are formed (33,48).  Aeetabulavia  Thus,  can r e a d i l y be e n u c l e a t e d by e x c i s i n g the r h i z o i d b e f o r e the c e l l  forms a  cap.  Aeetabulavia  I f immature  c e l l s are cut t o produce  a nucleate, rhizoid  p o r t i o n and an a n u c l e a t e s t a l k p o r t i o n , not o n l y can the n u c l e a t e r h i z o i d r e g e n e r a t e a complete s e v e r a l weeks and  new  p l a n t , but the a n u c l e a t e s t a l k can s u r v i v e f o r  c o n t i n u e t o grow.  even d i f f e r e n t i a t e a cap.  In some cases the a n u c l e a t e s t a l k  T h i s and o t h e r f i n d i n g s l e d Hammerling to p o s t u -  l a t e the e x i s t e n c e of "morphogenetic s u b s t a n c e s " which are produced n u c l e u s but may  will  by  be p r e s e n t i n the cytoplasm f o r l o n g p e r i o d s o f time  E x c i s i o n and i n t e r s p e c i f i c g r a f t i n g experiments  have shown t h a t  the  (33).  these  "morphogenetic s u b s t a n c e s " induce cap f o r m a t i o n and are n e c e s s a r y f o r d i f f e r e n t i a t i o n o f a cap w i t h s p e c i e s - s p e c i f i c morphology ( 3 3 ) . Within a single  Aeetabulavia  cell  are s e v e r a l m i l l i o n  chloroplasts.  4  The  number o f c h l o r o p l a s t s i n c r e a s e s  exponentially  of the c e l l , with the c h l o r o p l a s t doubling one week ( 5 5 ) . The c h l o r o p l a s t s i n c r e a s e a l s o , but i n t h i s case the d o u b l i n g The  time b e i n g a l i t t l e  T h i s was the f i r s t  longer  i n number i n a n u c l e a t e  than  fragments,  time i s about two weeks ( 5 5 ) .  presence o f DNA i n t h e c h l o r o p l a s t s o f  1963 (4,27).  d u r i n g normal growth  instance  Acetdbularva  was shown i n  i n which DNA c o u l d be d e f i n i t e l y  a t t r i b u t e d t o the c h l o r o p l a s t s w i t h o u t t h e p o s s i b i l i t y o f c o n t a m i n a t i o n by nuclear  DNA.  The presence o f DNA i n the c h l o r o p l a s t s l e a d s  to speculation  about i t s p o s s i b l e r o l e i n d i r e c t i n g the a c t i v i t i e s o f t h e c e l l .  Knowledge  of the amount o f DNA i n the c h l o r o p l a s t s and the presence o r absence o f repeated nucleotide tial  o f the c h l o r o p l a s t The  per  sequences can g i v e an i n d i c a t i o n o f the g e n e t i c  poten-  DNA.  s i t u a t i o n i s c o m p l i c a t e d by o b s e r v a t i o n s  t h a t t h e amount o f DNA  c h l o r o p l a s t i s v a r i a b l e ( 7 4 ) . U s i n g s e v e r a l t e c h n i q u e s , Woodcock and  Bogorad  (75) d e t e c t e d  the c h l o r o p l a s t s .  l a r g e , but v a r i a b l e , amounts o f DNA i n o n l y 35% o f  S i m i l a r l y , Green and B u r t o n (30) found DNA  w i t h o n l y 20-40% o f c h l o r o p l a s t s o s m o t i c a l l y  associated  shocked by the K l e i n s c h m i d t  t e c h n i q u e and observed w i t h the e l e c t r o n microscope. a t t r i b u t e t h i s to an i n t r a c h l o r o p l a s t a l n u c l e a s e .  Green and B u r t o n (30)  I n s p i t e o f these o b s e r -  v a t i o n s , however, e s t i m a t e s of the average amount o f DNA per c h l o r o p l a s t can be made. Using fluorometric  analyses,  Gibor and Izawa (27) e s t i m a t e d the  —16 amount o f DNA t o be 1 x 10 amount o f DNA i n a v i r u s .  g per c h l o r o p l a s t . Observation of  T h i s i s comparable t o the  ikcetabulaxrva  c h l o r o p l a s t DNA w i t h  the e l e c t r o n microscope has shown, however, t h a t a t l e a s t some c h l o r o p l a s t s  5  c o n t a i n much l a r g e r amounts o f DNA  (30,31,75).  e s t i m a t e o f G i b o r and Izawa i s too low.  T h i s suggests t h a t the  More r e c e n t l y , u s i n g  a scaled  down diphenylamine assay f o r DNA, Green determined t h a t the average amount of  DNA per c h l o r o p l a s t i s about 2.3 x 10  g (unpublished).  T b i s i s com-  p a r a b l e to the amount o f DNA i n t h e genomes o f a number o f b a c t e r i a l species, including  pneumoniae  and  Achromobacter  anitratus,  Staphylococcus  aureus  Streptococcus (3).  faecalis  Diplococcus  3  G i b o r and Izawa e s t i m a t e d t h e  amount o f DNA p e r c h l o r o p l a s t from the p r o t e i n to DNA r a t i o .  Their  calcu-  l a t i o n of the amount o f p r o t e i n p e r c h l o r o p l a s t was based on the assumption that proteins  comprise 20% o f the o r g a n e l l e s .  the number o f c h l o r o p l a s t s . tween the two r e s u l t s .  Perhaps t h i s accounts f o r t h e d i s c r e p a n c y be-  D e t e r m i n a t i o n o f the k i n e t i c c o m p l e x i t y o f t h e  c h l o r o p l a s t DNA should h e l p  to r e s o l v e  this.  A p r e r e q u i s i t e f o r the DNA r e n a t u r a t i o n double s t r a n d e d .  mediterranea  r e a c t i o n i s t h a t the DNA be  c h l o r o p l a s t DNA comes from e l e c t r o n microscopy (31,73) and  (32).  s t u d i e s o f c h l o r o p l a s t DNA which has been heat dena-  Thus, i t i s p o s s i b l e t o study the r e n a t u r a t i o n  c h l o r o p l a s t DNA.  Tbestudy the r e n a t u r a t i o n l y pure c h l o r o p l a s t DNA.  With  Acetabularia  chloroplast  i s o l a t i n g chloroplasts only  DNA.  k i n e t i c s , i t i s necessary to o b t a i n  Acetabularia,  relative-  c o n t a m i n a t i o n by n u c l e a r  can be r u l e d out by e x c i s i n g the r h i z o i d s , which c o n t a i n  but  k i n e t i c s o f the  The formula o f Wetmur and Davidson (74) can then be used  t o determine the k i n e t i c c o m p l e x i t y o f  difficult  Acetabularia  E v i d e n c e f o r the double strandedness o f  from buoyant d e n s i t y tured  Green d i d a c t u a l counts o f  DNA  the n u c l e i , and  from the s t a l k s o f immature c e l l s .  I t i s more  t o r u l e out the p o s s i b i l i t y o f c o n t a m i n a t i o n by m i t o c h o n d r i a l  the amount o f DNA i n a m i t o c h o n d r i o n appears t o be l e s s than i n a  DNA,  6 c h l o r o p l a s t (75).  Therefore, many mitochondria have to be present i n a  c h l o r o p l a s t preparation i n order to contribute a s i g n i f i c a n t amount of DNA.  Such an amount w i l l show up as a second UV absorbing band at p =  g/cm  i n a cesium c h l o r i d e d e n s i t y gradient (32).  1.714  Heilporn and Limbosch  (34) f i n d that the mitochondrial DNA renatures f a s t e r than the c h l o r o p l a s t DNA.  Therefore, mitochondrial DNA,  i f present, may be detected as a f a s t e r  renaturing component. Axenic c u l t u r e s of Acetabulavia  are d i f f i c u l t to maintain, so that  contamination with b a c t e r i a l DNA can be a problem. by t r e a t i n g the Acetabulavia  This can be minimized  c e l l s w i t h a n t i b i o t i c s before use, and by  s e l e c t i v e techniques during the c h l o r o p l a s t i s o l a t i o n .  The c h l o r o p l a s t pre-  parations can be monitored to determine the amount of b a c t e r i a l contamination. I f necessary, the c h l o r o p l a s t DNA can be separated from b a c t e r i a l DNA  by  c e n t r i f u g i n g i n a CsCl density gradient, provided that the b a c t e r i a l  DNA  has a d i f f e r e n t buoyant density than the c h l o r o p l a s t DNA.  Unfortunately,  there has been disagreement about the buoyant density of the c h l o r o p l a s t DNA as reported i n the l i t e r a t u r e (9, 26, 32, 34).  In the process of t h i s  3 research, t h i s problem was resolved. found f o r Acetabulavia  A buoyant density of 1.702  c h l o r o p l a s t DNA.  g/cm  was  This agrees q u i t e c l o s e l y with the  value given by Green et a l . (32) and Heilporn and Limbosch (34). Whatever the amount of DNA i n the c h l o r o p l a s t s , i t i s not very great and, thus, a major d i f f i c u l t y of t h i s research i s to o b t a i n large enough q u a n t i t i e s of DNA to study the r e n a t u r a t i o n k i n e t i c s . growth of Acetabulavia,  The r e l a t i v e l y slow  and the fact that each c e l l has to be handled i n -  d i v i d u a l l y to enucleate i t , are f u r t h e r l i m i t a t i o n s to preparing large amounts of Acetabulavia  c h l o r o p l a s t DNA.  7  MATERIALS AND METHODS  The f o l l o w i n g a b b r e v i a t i o n s a r e used: BSA, b o v i n e serum albumin; EDTA, d i s o d i u m 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 ; SDS, sodium d o d e c y l s u l p h a t e ; SSC, s t a n d a r d s a l i n e c i t r a t e pH 7.0); TES, N - t r i s Tris,  tris  (0.15 M NaCl p l u s 0.015 M sodium  (hydroxymethyl)methyl-2-aminoethane  citrate,  sulfonic  acid;  (hydroxymethyl)aminomethane.  Materials Reagent grade c h e m i c a l s were used throughout. from Pharmacia  (Uppsala, Sweden).  Miles Laboratories.  F i c o l l was purchased  Pentex brand BSA was o b t a i n e d from  TES was purchased from the N u t r i t i o n a l B i o c h e m i c a l  Company but c o n t a i n e d l a r g e amounts o f i m p u r i t i e s . p l i e s o f TES were o b t a i n e d from Calbiochem. l i q u i f i e d p h e n o l were used.  Therefore, l a t e r  sup-  M a l l i n c k r o d t phenol o r F i s h e r  P a n c r e a t i c r i b o n u c l e a s e and lysozyme were p u r -  chased from Worthington B i o c h e m i c a l Corp., w h i l e pronase and a-amylase were o b t a i n e d from Calbiochem.  R i b o n u c l e a s e T^ was purchased from the  N u t r i t i o n a l B i o c h e m i c a l Company.  Aquacide I I i s a p r o d u c t o f Calbiochem.  O p t i c a l grade C s C l was purchased from Schwarz B i o r e s e a r c h I n c . Treatment  o f c y s t s p r i o r to g e r m i n a t i o n :  Acetabulavia kill  cysts are r e s i s t a n t  growing c e l l s .  to k i l l i n g by some agents which  Thus, i n an attempt to o b t a i n a x e n i c c u l t u r e s ,  were t r e a t e d i n s e v e r a l ways.  will  cysts  A l l s o l u t i o n s and equipment were s t e r i l i z e d  b e f o r e use and s t e r i l e technique was used throughout the p r o c e d u r e s . R i p e caps which had been s t o r e d i n seawater i n the dark a t 10° f o r s e v e r a l months were washed w i t h seawater and c u t w i t h s c i s s o r s to r e l e a s e  8 the c y s t s .  ( I n some c a s e s , Shephard's  was used i n p l a c e of s e a w a t e r ) . cut caps were f i l t e r e d  artificial  seawater medium (57)  To s e p a r a t e c e l l d e b r i s from c y s t s , the  on 123 y mesh b o l t i n g s i l k , washed w i t h s e a water  and the c y s t s drawn through by g e n t l e s u c t i o n .  The c y s t s s e t t l e d t o the  bottom o f the f l a s k a f t e r one t o two minutes and the seawater was decanted. F r e s h seawater was added and the c o n t e n t s of the f l a s k shaken f o r one o r two minutes.  A f t e r the c y s t s s e t t l e d , the seawater was decanted.  washing procedure was r e p e a t e d f i v e or s i x t i m e s .  This  The c y s t s were then  t r e a t e d i n one o f the f o l l o w i n g ways: 1) The c y s t s were p l a c e d i n a s m a l l volume of 1% SDS i n d i s t i l l e d and the m i x t u r e a g i t a t e d f o r one hour w i t h a s m a l l magnetic bar.  water  stirring  The c y s t s were then a l l o w e d to s e t t l e and the SDS s o l u t i o n was  poured o f f .  F o l l o w i n g t h i s , the c y s t s were washed seven times w i t h  seawater, each time l e t t i n g the c y s t s s e t t l e and removing the seawater by d e c a n t a t i o n or by c a r e f u l l y withdrawing i t w i t h a P a s t e u r p i p e t t e . Then the c y s t s were p l a c e d i n 2 ml o f e n r i c h e d seawater medium o r a m i x t u r e o f e n r i c h e d seawater and Shephard's medium and l e f t at 20° t o germinate.  i n the l i g h t  ( E n r i c h e d seawater medium i s Shephard's medium i n  which seawater w i t h NaNO^ 0.04 g/1 and K^HPO^ 0.001 g/1 added, the m a c r o n u t r i e n t s a l t s o l u t i o n .  Micronutrient  v i t a m i n s a r e added as f o r Shephard's  replaces  s a l t s , NaHCO^ and  medium).  2) The c y s t s were t r e a t e d w i t h SDS as above, then p l a c e d i n n u t r i e n t Shephard's medium  (Shephard's medium w i t h g l u c o s e 0.6 g/1 and t r y p t o n e  0.6 g/1 added) w i t h p e n i c i l l i n 1 mg/ml, neomycin mycin 100 yg/ml i n the dark a t 2 0 ° .  100 yg/ml and s t r e p t o -  A f t e r two days, the c y s t s were  9 washed twice w i t h seawater and p l a c e d i n e n r i c h e d seawater i n t h e l i g h t to germinate. 3) The c y s t s (some w i t h , and some w i t h o u t , p r i o r treatment w i t h SDS as above), were t r e a t e d w i t h lysozyme 1 mg/ml i n 0.001 M T r i s , 0.001 M EDTA, pH 7.0, a t room temperature  f o r 20 minutes.  They were washed  twice w i t h seawater, then t r e a t e d w i t h 1% SDS i n d i s t i l l e d water f o r 15 minutes.  A f t e r b e i n g washed 5 t o 10 times w i t h seawater, the c y s t s  were p l a c e d i n 2 ml e n r i c h e d seawater i n the l i g h t to germinate.  In  some cases the c y s t s were t r e a t e d , as above, w i t h a n t i b i o t i c s i n t h e dark f o r two days b e f o r e b e i n g p l a c e d i n the l i g h t  to germinate.  4) A f t e r treatment w i t h SDS as above, the c y s t s were t r e a t e d w i t h 10% A r g y r o l i n seawater (27) f o r 20 minutes. were c o l l e c t e d on 25 u mesh b o l t i n g s i l k  Following t h i s ,  the c y s t s  (held i n a M i l l i p o r e  filter  a p p a r a t u s ) , washed w i t h 50 ml seawater, t r a n s f e r r e d t o a t e s t tube and washed t h r e e more times w i t h seawater.  Then they were put i n t o 2 ml  n u t r i e n t Shephard's medium w i t h p e n i c i l l i n 1 mg/ml and s t r e p t o m y c i n 100 iyig/ml i n t h e dark f o r two days.  F o l l o w i n g t h i s , t h e c y s t s were  washed t h r e e times w i t h seawater and p l a c e d i n 2 ml Shephard's medium i n the l i g h t  to germinate.  5) The c y s t s were t r e a t e d w i t h 10% A r g y r o l i n Shephard's medium f o r 20 minutes,  c o l l e c t e d on 25 ;u'mesh b o l t i n g s i l k and washed w i t h 150 ml  Shephard's medium.  They were then t r a n s f e r r e d t o a t e s t tube and washed  f i v e times w i t h Shephard's medium.  F o l l o w i n g t h i s , the c y s t s were  t r e a t e d w i t h 1% SDS i n d i s t i l l e d water f o r 15 minutes, times w i t h Shephard's medium.  then washed  F i n a l l y , the c y s t s were t r e a t e d w i t h  b i o t i c s as above b e f o r e b e i n g l e f t  i n the l i g h t  to germinate.  seven anti-  10  C u l t i v a t i o n and treatment  Aeetabulavia  o f algae  meditevvanea  c o n t a i n i n g 200 ml s t e r i l e c e l l s per f l a s k .  Shephard's medium  a lower l i g h t  flasks  (57), with approximately  200  They were grown a t 20°C and i l l u m i n a t e d w i t h 250-300 f t  candles of f l u o r e s c e n t l i g h t at  were c u l t i v a t e d i n 500 ml B e l l c o  f o r 12 hr/day.  i n t e n s i t y i n an attempt  Some of the c e l l s were grown  to i n h i b i t  cap f o r m a t i o n ( 4 8 ) .  For 2 to 5 days b e f o r e u s e , c e l l s were t r e a t e d w i t h one or more o f the f o l l o w i n g a n t i b i o t i c s , at the c o n c e n t r a t i o n s i n d i c a t e d :  penicillin  1 mg/ml, kanamycin 100 yg/ml, neomycin 100 yg/ml, s t r e p t o m y c i n 100 yg/ml or  chloramphenicol  used,  10-50 yg/ml.  When s t r e p t o m y c i n o r c h l o r a m p h e n i c o l were  the c e l l s were p l a c e d i n the dark to p r e v e n t damage to the c h l o r o -  plasts.  C e l l s were a l s o p l a c e d i n the dark to reduce  i n the c h l o r o p l a s t s and thus prevent t i o n procedure.  the amount of s t a r c h  c h l o r o p l a s t breakage d u r i n g t h e i s o l a -  The u s u a l regime was to wash the c e l l s  two o r t h r e e  and p l a c e them i n f r e s h medium w i t h neomycin f o r two days.  times  Then the c e l l s  were washed twice and p l a c e d i n f r e s h medium w i t h p e n i c i l l i n and kanamycin or  s t r e p t o m y c i n f o r two days i n the dark.  pare  The c e l l s were then used  to p r e -  chloroplasts.  I s o l a t i o n of Chloroplasts: Sterile sterilized  technique was used throughout.  i n an a u t o c l a v e o r oven.  through a 0.22 y M i l l i p o r e C e l l s two to f i v e  Equipment and glassware were  S o l u t i o n s were s t e r i l i z e d by passage  filter.  cm i n l e n g t h were used, p r i o r t o cap f o r m a t i o n .  a few c a s e s , c e l l s w i t h cap i n i t i a l s  l e s s than 1.5 mm i n diameter were  In used.  11  The c e l l s were washed t h r e e times and each c e l l e n u c l e a t e d by c u t t i n g o f f the r h i z o i d a l o n g w i t h 2 t o 3 mm o f the s t a l k , u s i n g i r i d e c t o m y scissors.  In a few i n s t a n c e s , the n u c l e a t e fragments  p l a c e d i n f r e s h medium to r e g e n e r a t e new s t a l k s . of one o r two hours i n dim l i g h t ,  were c o l l e c t e d and  A f t e r a recovery period  the s t a l k s were c o l l e c t e d and used.  In  p r e l i m i n a r y p r e p a r a t i o n s , Shephard's method was used to i s o l a t e the c h l o r o plasts  ( 5 9 ) . T h i s was m o d i f i e d s l i g h t l y f o r l a t e r p r e p a r a t i o n s . A l l  o p e r a t i o n s were c a r r i e d out a t 0-4°. bucket r o t o r of a S o r v a l l R2B Approximately  C e n t r i f u g i n g was done i n the swinging  centrifuge.  2 g wet weight  of e n u c l e a t e d c e l l s were s c i s s o r minced  i n 3 ml o f a medium c o n s i s t i n g o f 0.6 M m a n n i t o l , 0.1% BSA, 0.001 M EDTA, 0.1 M TES and 0.001 M d i t h i o t h r e i t o l , pH 7.8 filtered  (H medium).  The s l u r r y was  through 25 ymesh b o l t i n g s i l k under g e n t l e s u c t i o n and washed  through w i t h a p p r o x i m a t e l y 3 ml o f a medium s i m i l a r to H but c o n t a i n i n g 0.01 M TES (W medium).  C e l l w a l l d e b r i s was r e t a i n e d by the b o l t i n g s i l k , w h i l e  the c h l o r o p l a s t s and c y t o p l a s m i c content passed  through.  The c h l o r o p l a s t  s u s p e n s i o n was l a y e r e d onto 4 ml of W medium w i t h 2% F i c o l l added medium) and c e n t r i f u g e d f o r 15 min a t 650 x g. was d i s c a r d e d .  The p a l e green  (WF  supernatant  The c h l o r o p l a s t p e l l e t was suspended i n 4 ml W medium, l a y -  ered over 4 ml WF medium and c e n t r i f u g e d a t a p p r o x i m a t e l y 90 x g f o r 5 minutes.  The v e r y s m a l l green and white p e l l e t was d i s c a r d e d and the green  supernatant was passed through a 5 y Nucleopore aggregates  f i l t e r t o break up c h l o r o p l a s t  and shear o f f tags o f cytoplasm a d h e r i n g to the c h l o r o p l a s t s .  T h i s step a l s o removed a s i g n i f i c a n t number of b a c t e r i a .  The f i l t r a t e  t a i n i n g the c h l o r o p l a s t s was then c e n t r i f u g e d a t 650 x g f o r 15 minutes and the p a l e green supernatant d i s c a r d e d .  The c h l o r o p l a s t p e l l e t  was  con-  12  suspended  i n 4 ml W medium, l a y e r e d o v e r 4 ml WF medium and c e n t r i f u g e d  at 650 x g f o r 15 minutes.  ( T h i s step was sometimes o m i t t e d ) .  green supernatant was d i s c a r d e d .  The p a l e  The c h l o r o p l a s t p e l l e t was suspended i n  5 ml o f a medium c o n t a i n i n g 0.6 M m a n n i t o l , 0.1% BSA, 0.025 M TES, 0.01 M KC1, 5 mM M g C l  2  and 0.5 mM KH^PO^, pH 7.8 (A medium) and c e n t r i f u g e d  at 650 x g f o r 15 minutes. discarded  The v e r y p a l e green o r c l e a r s u p e r n a t a n t was  and the c h l o r o p l a s t p e l l e t  on the amount o f s t a r t i n g and suspended  resuspended  i n A medium.  Depending  m a t e r i a l , from one t o f o u r p e l l e t s were combined  i n a f i n a l volume o f 6.6 t o 16.6 ml o f A medium.  After  thorough m i x i n g o f the c h l o r o p l a s t s , 0.6 ml was taken to assay f o r b a c t e r i a l c o n t a m i n a t i o n and the remaining c h l o r o p l a s t s u s p e n s i o n was c e n t r i f u g e d a t 650 x g f o r 15 minutes.  The supernantant was d i s c a r d e d  c o n t a i n i n g 90-95% i n t a c t  c h l o r o p l a s t s , was used f o r the p r e p a r a t i o n  I n a few p r e l i m i n a r y p r e p a r a t i o n s ,  and the p e l l e t  i n s t e a d o f suspending  o f DNA.  the c h l o r o -  p l a s t s i n A medium i n the f i n a l s t e p s , the c h l o r o p l a s t p e l l e t was  suspended  i n 4 ml W medium and l a y e r e d over 4 ml 0.8 M s u c r o s e i n 0.005 M EDTA, 0.01 M TES, 0.001 M d i t h i o t h r e i t o l , pH 7.8. 650 x g f o r 15 minutes.  The s u s p e n s i o n was c e n t r i f u g e d a t  Sometimes t h i s was not adequate  t o p e l l e t the  c h l o r o p l a s t s , i n which case c e n t r i f u g a t i o n was repeated a t 1000 x g. c h l o r o p l a s t p e l l e t was then suspended  i n 5 ml 0.4 M s u c r o s e b u f f e r e d as  above, and 0.7 ml taken f o r c o n t a m i n a t i o n check. was c e n t r i f u g e d a t 650 x g f o r 15 minutes for preparation  Preparation  The  The nemaining  suspension  and the c h l o r o p l a s t p e l l e t  used  o f DNA.  o f c h l o r o p l a s t DNA  In p r e l i m i n a r y  experiments, DNA was prepared by a s l i g h t  of the phenol-pH9-RNases method o f M i u r a (46).  modification  The c h l o r o p l a s t p e l l e t  from  13  2-4  buffer min  Acetabutavia  g wet weight of  (0.1 M T r i s , 1% SDS,  f o r l y s i s to o c c u r .  b u f f e r was  added and  c e l l s was  suspended i n 2-3  0.1 M N a C l , pH 9.0)  and l e f t  ml t r i s - S D S -  on i c e f o r  10-15  An e q u a l volume o f phenol s a t u r a t e d w i t h t r i s - S D S  the mixture  shaken i n the c o l d f o r 20 min.  Layers  were s e p a r a t e d by c e n t r i f u g a t i o n and the c l e a r , upper, aqueous l a y e r c a r e f u l l y pipetted off.  S i n c e t h e r e was  e t h a n o l , the aqueous l a y e r was in  of  the p o o l e d samples was  cubated  DNA  DNA  to two h o u r s .  yg/ml was  added and  model L2B  The  s o l u t i o n was  a d j u s t e d to a d e n s i t y o f 1.700  punctured  g/cm  w i t h a 27 gauge needle and 4 drop d i l u t e d w i t h 0.5  Peak f r a c t i o n s c o n t a i n i n g DNA  used  added and  i n the  50°  the i n -  then d i a l y s e d a g a i n s t II.  The  by the a d d i t i o n o f  ml 0.1  x SSC  o f p = 1.702  g/cm  f r a c t i o n s were c o l l e c t e d .  and the OD^..^ monitored. 260nm  were p o o l e d and  dialysed  cold.  l a t e r experiments, to p r e p a r e DNA  suspended i n 1.25  the  The bottom o f the c e n t r i f u g e  3  was  solution i n -  i n the 50 T i r o t o r of a Beckman  u l t r a c e n t r i f u g e f o r 61 h r a t 20°.  Each f r a c t i o n was  In  volume  the s o l u t i o n i n c u b a t e d at  An a d d i t i o n a l 50 yg pronase/ml was  s o l i d C s C l and c e n t r i f u g e d at 42000 rpm  i n t o SSC  The  (2-3 changes) i n the c o l d and c o n c e n t r a t e d w i t h Aquacide  s o l u t i o n was  tube was  I I and t h e DNA  pooled  Pronase ( f r e e o f d e o x y r i b o n u c l e a s e by  c u b a t i o n c o n t i n u e d f o r 2 to 3 hours. 2 1 SSC  (3 changes)  from s e v e r a l p r e p a r a t i o n s was  reduced w i t h Aquacide  with  at 37° w i t h r i b o n u c l e a s e T^ 25-100 u n i t s / m l and p a n c r e a t i c r i b o -  method of S t e r n (62) 100 one  x SSC  (4°) w i t h a drop o f c h l o r o f o r m .  n u c l e a s e 50 y§/ml f o r 30-45 min.  for  to be p r e c i p i t a t e d  d i a l y z e d a g a i n s t 2 1 0.1  the c o l d f o r 24 t o 48 hours.  and s t o r e d i n the r e f r i g e r a t o r  not enough DNA  a s l i g h t m o d i f i c a t i o n o f Marmur's method  from the c h l o r o p l a s t s .  ml 0.15  The  (44)  chloroplast pellet  M N a d * 0.1 M EDTA, pH 8 and 0.1  ml 25%  SDS  was was  14  added. ice.  The  m i x t u r e was  Following  the a d d i t i o n o f 0.33  form: isoamyl a l c o h o l cold  heated t o 60°  (24:1  f o r 20 minutes.  The  V/V)  was  f o r 10 minutes and ml  added and  dialysed against  hours. DNA  After dialysis  the DNA  ribonuclease  f o r 30 minutes, and added and  s o l u t i o n was  pronase 50  yg/ml was  hours.  The  2 1 SSC  i n the  100  yg/ml and  and  the DNA  ribonucleases,  s o l u t i o n was  2 1 SSC  a-amylase and  25-50  Pronase 100  A f t e r one  i n the  to two  3-1/2  dark.  pronase, as above, was  clarified  A s m a l l w h i t e p e l l e t was  d i a l y s e d i n t o SSC  or 0.1  x  against diaTreat-  repeated  After further  s o l u t i o n was  yg/ml  hours,  overnight  c o l d i n the  SSC.  37°  units/ml  c o n t i n u e d f o r 2 to  again d i a l y s e d against  c l e a r s u p e r n a t a n t was  24  (-20°).  c o l d i n the dark, c o n c e n t r a t e d w i t h Aquacide I I , and  c e n t r i f u g i n g at 18000 x g f o r 10 min. the  dark f o r  freezer  T^  then d i a l y s e d  t i o n of the volume w i t h Aquacide I I , the DNA  and  The  p o o l e d , then t r e a t e d at  the i n c u b a t i o n  l y s e d a f u r t h e r 6 to 18 h r a g a i n s t ment w i t h the  i n the  ribonuclease  c h i l l e d on i c e and  the  c a r e f u l l y withdrawn  c o l d i n the  at 50°.  on  chloro-  centrifugation.  yg/ml f o r 15 minutes.  c a r r i e d out  added and  s o l u t i o n was  was  stored  thawed and  a-amylase 5-10  the i n c u b a t i o n  the DNA  (2 changes) i n the  from s e v e r a l p r e p a r a t i o n s was  with pancreatic  was  2 1 SSC  volume o f  the m i x t u r e shaken i n  l a y e r s were s e p a r a t e d by  c l e a r , upper, aqueous l a y e r c o n t a i n i n g and  5 M NaClO^, one  then c h i l l e d  reducby  discarded  SSC.  Monitoring f o r b a c t e r i a l contamination To of the  check the e f f e c t i v e n e s s  of various  f i n a l wash, a l o n g w i t h a few  treatments of c y s t s ,  c y s t s , were p l a t e d  agar ( n u t r i e n t Shephard's medium s o l i d i f i e d w i t h 1.5% agar o v e r l a y and  and  i n c u b a t e d at 20°.  contaminants, when p r e s e n t , were  A f t e r one  aliquots  on n u t r i e n t  Shephard's  agar) w i t h a s o f t  week c o l o n i e s were counted  characterized.  15  Occasionally,  individual  Acetabulavia  c e l l s were observed w i t h a  phase microscope to check f o r the presence o f b a c t e r i a and o t h e r p r o t i s t s . At  s e v e r a l times d u r i n g  the growth phase,  Acetabulavia  c e l l s were p l a t e d  on n u t r i e n t Shephard's agar a l o n g w i t h a s m a l l amount o f the medium i n which they were growing. and  e x t e n t o f b a c t e r i a l growth was noted. To  the  P l a t e s were i n c u b a t e d a t 20° and the n a t u r e  check f o r c o n t a m i n a t i o n i n c h l o r o p l a s t p r e p a r a t i o n s ,  f i n a l c h l o r o p l a s t s u s p e n s i o n were added t o n u t r i e n t -1  to make f i n a l d i l u t i o n s o f 10  -2 , 10  aliquots of  Shephard's medium  -3 and 10  .  F i v e tubes o f each  dilu-  t i o n were i n c u b a t e d a t 20° and the number o f samples o f each d i l u t i o n which became t u r b i d a f t e r one week were counted.  Most p r o b a b l e numbers o f b a c -  t e r i a were e s t i m a t e d from the t a b l e g i v e n by C o l l i n s ( 1 7 ) . I n a d d i t i o n to t h i s , a l i q u o t s o f each d i l u t i o n were p l a t e d on n u t r i e n t Shephard's agar. Colonies week. 10 and  were counted and c h a r a c t e r i z e d  Chloroplast  a f t e r incubation  a t 20° f o r one  numbers were determined by c o u n t i n g two a l i q u o t s o f a  d i l u t i o n i n a hemacytometer.  The r a t i o o f b a c t e r i a t o c h l o r o p l a s t s  thus the amount o f DNA b e i n g c o n t r i b u t e d  by b a c t e r i a c o u l d  then be  estimated. I s o l a t i o n o f DNA  from b a c t e r i a l contaminants  B a c t e r i a were grown i n n u t r i e n t Shephard's medium o v e r n i g h t s h a k i n g a t 25°. according  C e l l s were h a r v e s t e d by c e n t r i f u g a t i o n and DNA  with extracted  to the phenol-pH9-RNases method o f M i u r a ( 4 6 ) . F r e e z i n g and  thawing was not n e c e s s a r y to l y s e the c e l l s , but i n some cases pronase ( f r e e o f d e o x y r i b o n u c l e a s e (62) 100 yg/ml was used t o d i s r u p t  filaments.  16 Cesium c h l o r i d e d e n s i t y g r a d i e n t s About 0.8  ml of the s o l u t i o n c o n t a i n i n g l-5ug DNA/ml was  adjusted  3 to  a d e n s i t y of 1.70  g/cm  by the a d d i t i o n o f s o l i d  Micrococcus  Q?C1.  3 lysodeikticus The AN-D  (p = 1.731  DNA  s o l u t i o n was  g/cm  ) was  c e n t r i f u g e d at 44,000 rpm  r o t o r o f a Spinco Model E at 2 0 ° .  h r and  added as a d e n s i t y r e f e r e n c e .  the n e g a t i v e s  i n a 12 mm  K e l - F c e l l i n the  Photographs were taken a f t e r  scanned w i t h a J o y c e - L o e b l m i c r o d e n s i t o m e t e r .  20 Buoy-  ant d e n s i t i e s were c a l c u l a t e d a c c o r d i n g to Mandel et^ a l . (43) , without correction for pressure  affects.  E l e c t r o n microscopy C h l o r o p l a s t s were f i x e d i n 2.5% b u f f e r , pH 6.8,  glutaraldehyde  and p o s t - f i x e d i n 1% 0s0^  i n sodium  cacodylate  i n cacodylate b u f f e r .  After  h y d r a t i n g w i t h an e t h a n o l s e r i e s , the c h l o r o p l a s t s were i n f i l t r a t e d and  de-  with,  embedded i n , Spurcls medium (60) i n g e l a t i n c a p s u l e s ; cured i n a vacuum  oven at 70° f o r 10 hours and  t h i n sectioned.  S i l v e r s e c t i o n s were c o l l e c t e d  on 200 mesh copper g r i d s and s t a i n e d w i t h u r a n y l a c e t a t e f o l l o w e d by l e a d citrate.  S e c t i o n s were examined w i t h an H i t a c h i HU  11-A  o r HS-7S e l e c t r o n  microscope. P r e p a r a t i o n of  E. coli  E. coli  K 12 was  partment, U.B.C. extract  DNA  was  obtained  7.5,  at 30° or 37° w i t h shaking  e x t r a c t e d by  t i o n a l p u r i f i c a t i o n steps. 50 yg/ml and  from Dr. R. Warren of the M i c r o b i o l o g y  C e l l s were grown i n n u t r i e n t b r o t h  ( D i f c o ) , pH  fugation.  DNA  r i b o n u c l e a s e T,  the method of M i u r a  F o l l o w i n g treatment  ( D i f c o ) w i t h 5% and h a r v e s t e d by  Deyeast centri-  (46) , w i t h s e v e r a l a d d i -  with pancreatic ribonuclease  25 u n i t s / m l at 37° f o r 30 m i n u t e s , the  DNA  17 i n SSC 50°  was  i n c u b a t e d w i t h pronase ( d e o x y r i b o n u c l e a s e f r e e ) 100  f o r one  to two  f o r 2 to 3 hours. added.  DNA  dissolved justed  hr.  Pronase 50  The  s o l u t i o n was  i n 0.1  ates the DNA  One  DNA  (35).  Ralph ( 7 ) , was  volume c o l d 2.5  Kirby's  p r e c i p i t a t e was (4°)  two  M N a C l , pH  7.5,  p h e n o l f o r 15 min  8.1,  The  layers.  8, and  one  x SSC  and  the  The  0.025 M  volume c o l d f o r 3 minutes  c l e a r upper l a y -  s o l u t i o n stored  of Dr.  in  R. M i l l e r o f the  phage p a r t i c l e s i n 0.5  m i x t u r e was The  o f f and  dialysed against  24 h r s .  The  s o l u t i o n was  chloroform.  des-  The  the  DNA  pipetted  DNA  step separ-  containing  ml  o f 0.01  were shaken w i t h an equal volume of water  at 4 ° .  to s e p a r a t e the  drop of  This  one  chloroform.  o f b a c t e r i o p h a g e T4  The  ad-  volumes o f c o l d e t h a n o l added to i t .  d i s s o l v e d i n 0.1  department, U.B.C.  and  used to remove p o l y s a c c h a r i d e s .  P u r i f i e d phage T4 p a r t i c l e s were a g i f t biology  ethanol  s o l u t i o n was  the mixture shaken v i g o r o u s l y  w i t h a drop of  continued  two-phase method, as  M phosphate b u f f e r , pH  c a r e f u l l y withdrawn and  Preparation  min  The  A f t e r c e n t r i f u g i n g at 12,000 x g f o r 5 minutes, the  refrigerator  0.15  6.0.  added to p r e c i p i t a t e the DNA.  2-methoxyethanol were added and  e r was  treatment  2 volumes of c o l d  d i s s o l v e d i n 0.025 M T r i s - H C l b u f f e r , pH  at 4 ° .  the  a d d i t i o n o f 3 M sodium a c e t a t e and  from r i b o n u c l e o t i d e s  c r i b e d by Bellamy and  NaCl.  c h i l l e d and  M sodium a c e t a t e b u f f e r , pH  to 0.13 M sodium a c e t a t e by  was  added and  f i b e r s were c o l l e c t e d on a g l a s s r o d , washed i n e t h a n o l  volume o f e t h o x y e t h a n o l was  DNA  yg/ml was  yg/ml at  centrifuged  then s t o r e d  Tris,  saturated  at 2000 x g f o r  c l e a r upper aqueous l a y e r was 2 1 of SSC  M  Micro-  (2 changes) i n the  10  carefully cold  for  i n the r e f r i g e r a t o r (4°) w i t h a  18 Thermal d e n a t u r a t i o n p r o f i l e s and d e t e r m i n a t i o n o f Tm S o l u t i o n s o f DNA i n SSC o r 0.1 x SSC were degassed by e v a c u a t i o n i n a vacuum d e s s i c a t o r .  S p e c t r o s i l semi-micro c u v e t t e s f i t t e d w i t h t e f l o n  s t o p p e r s were used and the QJ^Onm a u t o m a t i c a l l y r e c o r d e d w i t h a G i l f o r d 2400 spectrophotometer.  The temperature i n the chamber was i n c r e a s e d r a -  p i d l y to about 50° and them a t a r a t e of 1° p e r 4 min u n t i l the onset o f m e l t i n g . A f t e r t h a t p o i n t , the  temperature was i n c r e a s e d 1° p e r 10 min  u n t i l maximum h y p e r c h r o m i c i t y was reached.  Absorbance measurements were  c o r r e c t e d f o r t h e r m a l expansion of the s o l v e n t and the Tm determined from the p l o t o f r e l a t i v e absorbance (A,j,/A2^o) v e r s u s temperature (42) . p l e of  E. coli-  A sam-  K 12 DNA was i n c l u d e d as a r e f e r e n c e d u r i n g each experiment.  R e n a t u r a t i o n o f DNA The DNA samples were d i a l y s e d i n t o the a p p r o p r i a t e s o l v e n t and an a l i q u o t of the d i a l y s i s s o l u t i o n was u s e d ^ f o r the absorbance r e f e r e n c e . DNA was sheared by passage through a 27 gauge n e e d l e , u s i n g a 2 ml s y r i n g e and a p p l y i n g maximum p r e s s u r e by hand.  DNA s o l u t i o n s a t c o n c e n t r a t i o n s o f  6 to 20 yg/ml were degassed by e v a c u a t i o n i n a vacuum d e s s i c a t o r .  The r e -  n a t u r a t i o n r e a c t i o n was f o l l o w e d o p t i c a l l y , w i t h the DNA s o l u t i o n s  contained  i n S p e c t r o s i l semi-micro c u v e t t e s f i t t e d w i t h t e f l o n s t o p p e r s and the absorbance a t 260 nm b e i n g measured i n a G i l f o r d 2400 a u t o m a t i c r e c o r d i n g spectrophotometer.  I n any experiment, two o r t h r e e samples were r u n s i m u l -  taneously. In one experiment, a f t e r d e t e r m i n i n g the  0^260  o f the n a t i v e DNA, the  DNA was denatured by adding one p a r t 1.0' M NaOH to 8 p a r t s o f the DNA s o l u t i o n i n 4.5 x SSC.  A f t e r 10 minutes a t room temperature t h e 0 D  9ftf)  of  19 the  denatured DNA  was  read.  The s o l u t i o n was  then n e u t r a l i z e d by adding  one p a r t 2 M NaH^PO^, mixed w e l l , and absorbance r e a d i n g s begun immediately w h i l e the s o l u t i o n e q u i l i b r a t e d to the r e n a t u r a t i o n temperature. r e n a t u r e d at 70 + 1.0°C.  T h i s i s (Tm-28?°  —  T4 DNA  for  m  at t h i s i o n i c strength (74).  The Tm  E. coll  for  DNA  Acetabulavia  and  DNA  was  (Tm-22)° f o r  chloroplast  DNA  has not been determined i n a s o l v e n t of t h i s i o n i c s t r e n g t h , but from  the  base compositions the Tm o f the c h l o r o p l a s t DNA  of E. coll In  DNA and T4 DNA.  another experiment, DNA  temperature was In DNA,  minutes.  to  then lowered to 6 3 + 0 . 5 °  i n 0.1 x SSC was  and the DNA  The  a l l o w e d to r e n a t u r e .  heated on a b o i l i n g water b a t h f o r 12 to 15  U s i n g a warm p i p e t t e , 1.625  ml 20 x SSC  65°.  i n SSC was melted as d e s c r i b e d above.  o t h e r experiments, a f t e r d e t e r m i n i n g the absorbance o f the n a t i v e  the DNA  0.175  s h o u l d be between t h o s e  ml hot denatured DNA  ( f i n a l c o n c e n t r a t i o n 2 x SSC)  The s o l u t i o n s were mixed  was  added t o  i n a cuvette equilibrated  and absorbance r e a d i n g s begun immediately.  Since the temperature r e c o r d e d on the c h a r t d i f f e r e d s l i g h t l y from the a c t u a l temperature of the s o l u t i o n s i n the c u v e t t e s , an a d d i t i o n a l c u v e t t e c o n t a i n ing  2 x SSC was  included.  w i t h a Tele-Thermometer  The  temperature o f t h i s s o l u t i o n was  monitored  ( Y e l l o w S p r i n g s Instrument Company) and used to  c a l i b r a t e the temperature r e c o r d e d on the c h a r t . Absorbance of  the DNA  v a l u e s were c o r r e c t e d as n e c e s s a r y f o r changes  solution.  i n the volume  C o r r e c t i o n s f o r thermal expansion o f the s o l v e n t were  a l s o made. The of  c h l o r o p l a s t DNA  s o l u t i o n , i n p a r t i c u l a r , c o n t a i n e d a contaminant  unknown n a t u r e which had a c o n s i d e r a b l e absorbance at 260 nm.  For t h i s  20 reason, the c o n c e n t r a t i o n of DNA micro  i n each sample was  determined  assay u s i n g Burton's m o d i f i c a t i o n of the diphenylamine  The percentage  o f the 0^250  w  n  i  c  n  w  a  due  s  DNA  t o  s  w a  by  a  assay  (15).  then c a l c u l a t e d ,  and  a l l a a b s o r b a n c e r e a d i n g s c o r r e c t e d a c c o r d i n g to t h i s v a l u e . The used  f o l l o w i n g equations g i v e n by Wetmureand Davidson  t o determine  A„ - A 6 °° A - A  =  the  (74) were then  :  k„ P_ 2 T 2  and P_ T  1  = 1.47  x 10  _ 4  A  00  mole  1  _ 1  00 where  A  i s the absorbance of n a t i v e  DNA;  00 A  o  i s the maximum absorbance of denatured  A  i s the absorbance of the p a r t i a l l y r e n a t u r e d DNA  t  i s the time, i n seconds,  P^  i s the t o t a l DNA  and  i s the second  order r e n a t u r a t i o n r a t e constant.  s t r e n g t h of the s o l v e n t , a l l  (11).  (39,  M,  u s i n g v a l u e s of r e l a t i v e  values f o r  to 50% G+C,  ionic  to a s o l v e n t monova-  Base c o m p o s i t i o n of the DNA  renaturation rate, therefore were n o r m a l i z e d  i s i n f l u e n c e d by the  v a l u e s were n o r m a l i z e d  l e n t c a t i o n c o n c e n t r a t i o n of 1.0 r a t e s g i v e n by B r i t t e n  at time t ;  phosphate c o n c e n t r a t i o n ;  S i n c e the r a t e of r e n a t u r a t i o n of the DNA  T4 DNA  DNA;  Aeetabulavia  reassociation  also affects i t s c h l o r o p l a s t DNA  u s i n g d a t a g i v e n by Wetmur and  and  Davidson  74).  D e t e r m i n a t i o n of a l k a l i n e s e d i m e n t a t i o n Band v e l o c i t y s e d i m e n t a t i o n was c o e f f i c i e n t of denatured 28,000 o r 30,000 rpm  DNA  (63,69).  i n the AN-D  coefficients  used  t o determine  the  Sedimentation was  sedimentation  c a r r i e d out at  r o t o r of a Spinco Model E u l t r a c e n t r i f u g e ,  21  at a temperature min  o f 22° to 2 5 ° . Photographs were taken a t 8 min or 16  i n t e r v a l s and the n e g a t i v e s scanned w i t h a J o y c e - L e o b l m i c r o d e n s i t o -  meter. About 25 ,yil o f a DNA s o l u t i o n i n 0.1 x SSC ( 0 D the sample w e l l  -0.4) was used i n  2 6 Q  o f a 12 mm K e l - F band forming c e n t e r p i e c e .  I n some cases  0.5 ml o f 0.9 M NaCl, 0.1 M NaOH was used as the b u l k s o l u t i o n and t h e a l k a l i n e s e d i m e n t a t i o n c o e f f i c i e n t (S??"^) determined 20 ,w c a s e s , the DNA was heat denatured  directly.  I n other  on a b o i l i n g water b a t h and q u i c k c o o l e d  on i c e b e f o r e b e i n g loaded i n t o the sample w e l l .  The b u l k s o l u t i o n was  1.0 M N a C l , 0.05 M sodium c i t r a t e and the a l k a l i n e s e d i m e n t a t i o n c o e f f i c i e n t was s t i p u l a t e d from t h e f o l l o w i n g  r e l a t i o n s h i p d e r i v e d from e q u a t i o n s  by S t u d i e r ( 6 3 ) : log S ^  1 3  , w  =  0.400 0.549  ( l o g PH7,denatured _ S  '  W  l  Q  g  0  Q  1  Q  5  )  +  l  o  g  0  .  0 5  28  given  22 RESULTS  Attempts t o o b t a i n axenic c u l t u r e s o f  Aeetabulavia  Washing  Aeetabulavia  c y s t s w i t h seawater, a l o n e , was important i n  r e d u c i n g the amount of b a c t e r i a l c o n t a m i n a t i o n . medium used of  P l a t i n g o f 0.1 ml o f  f o r the f i r s t wash r e s u l t e d i n heavy, c o n f l u e n t white  bacteria.  growth  A f t e r the f i f t h wash, 0.1 ml of washing medium gave r i s e t o  300-400 b a c t e r i a l c o l o n i e s .  Treatment o f c y s t s w i t h SDS was v e r y  t i v e a g a i n s t b a c t e r i a which produced a g a i n s t the contaminants  effec-  white c o l o n i e s , but n o t as e f f e c t i v e  which gave r i s e to y e l l o w c o l o n i e s .  Although  a x e n i c c u l t u r e s d i d not r e s u l t , the degree o f c o n t a m i n a t i o n was s l i g h t , w i t h 0.1 ml o f the f i n a l washing medium g i v i n g r i s e to 5-10 b a c t e r i a l colonies.  Lysozyme was n o t as e f f e c t i v e as SDS i n e l i m i n a t i n g the white  contaminants,  b u t was more e f f e c t i v e a g a i n s t the y e l l o w  contaminants.  Treatment o f c y s t s w i t h lysozyme f o l l o w e d by SDS was more e f f e c t i v e e i t h e r of these agents were n o t o b t a i n e d . of  than  a l o n e , although completely a x e n i c c u l t u r e s s t i l l  A n t i b i o t i c treatment was h e l p f u l i n r e d u c i n g the number  b a c t e r i a , b u t was n o t s u f f i c i e n t  to produce a x e n i c  A r g y r o l was the most e f f e c t i v e agent  cultures.  for eliminating bacterial  minants but i t was a l s o t h e most damaging t o  Aeetabulavia  cysts.  conta-  Cysts  t r e a t e d n t ' w i t h a r g y r o l a f t e r b e i n g t r e a t e d w i t h SDS f a i l e d to germinate, and  died.  When c y s t s were t r e a t e d w i t h a r g y r o l a l o n e , o r when SDS treatment  f o l l o w e d the treatment, w i t h a r g y r o l , however, the c y s t s s u r v i v e d and g e r minated. SDS if  T h i s suggests  t h a t c y s t s which have been p r e v i o u s l y t r e a t e d w i t h  may be more permeable to a r g y r o l and t h a t a r g y r o l i s t o x i c t o t h e c y s t s i t p e n e t r a t e s them.  Some axenic c u l t u r e s o f  Aeetabulavia  were o b t a i n e d  23 by  t r e a t i n g the c y s t s w i t h a r g y r o l , but  i n most cases c o n t a m i n a t i o n  a c c i d e n t a l l y introduced-: i n t o the c u l t u r e s b e f o r e were f u l l y grown.  When  Acetabulavia  did  not  due  to a l a c k of some m e t a b o l i t e  Acetabulavia  the  cells  c u l t u r e s d i d remain a x e n i c ,  appear to grow as w e l l as non-axenic c u l t u r e s . which i s s u p p l i e d by  was  the  Whether t h i s  cells was  the b a c t e r i a i n I |  contaminated c u l t u r e s , or whether r e s i d u a l t r a c e s o f Ag i n h i b i t e d growth o f the At one  Acetabulavia  c e l l s , was  not  p o i n t , h a l f of the s t o c k c u l t u r e s o f  from the  argyrol  investigated.  Acetabulavia  became con-  taminated w i t h  Nanachloves.  P r o b a b l y the c y s t s were contamined w i t h  chloves  germination.  I t was  before  a r i s i n g from c y s t s which had growing i n w i t h them, but which had  been t r e a t e d w i t h SDS  Acetabulavia  These are l i s t e d i n t a b l e I , t o g e t h e r  s i t i e s o f t h e i r DNA.  The  first  i n the  density of p l a s t DNA  Acetabulavia  f o u r types o f b a c t e r i a l i s t e d i n the  the  c h l o r o p l a s t DNA,  i t was  p o s s i b l e to s e p a r a t e  by means o f a p r e p a r a t i v e  CsCl gradient.  chloroAl-  done i n p r e l i m i n a r y experiments, the amount o f b a c t e r i a l  c o n t a m i n a t i o n was later  Since  table  from these contaminants d i f f e r from the buoyant  from b a c t e r i a l DNA  though t h i s was  chloroplast  w i t h the buoyant den-  were the ones most commonly found i n c h l o r o p l a s t p r e p a r a t i o n s . bjuoyant d e n s i t i e s of DNA  cysts  preparations  types o f b a c t e r i a l contaminants were p r e s e n t  preparations.  'Nanachloves  Nanachloves.  were contaminated w i t h  B a c t e r i a l contamination i n c h l o r o p l a s t  cells  d i d not have any  a l l the c u l t u r e s a r i s i n g from  not been t r e a t e d w i t h SDS  Several  Acetabulavia  observed t h a t the  Nana-  low  preparations.  enough t h a t t h i s was  not  considered  necessary with  24 Table I :  B a c t e r i a l contaminants of c h l o r o p l a s t  preparations  Macroscopic Appearance  Microscopic Appearance  Gram Reaction  Buoyant Density  a) s h i n y , w h i t e , smooth, domed colony with e n t i r e margin  motile rod  negative  1.716 g/cm"  b) creamy-white, s h i n y , domed c o l o n y w i t h e n t i r e margin  motile; short, f a t rods  positive  1.718 g/cm"  c) s m a l l s h i n y , g o l d en-yellow, f l a t colony. Colonies have " f r i e d - e g g " appearance on o l d plates  non-motile; t h i n rods  positive  1.699 g/cm"  d) v e r y s m a l l , s h i n y , golden-yellow colony  non-motile; l o n g , filamentous rods, s t r a i g h t and curved;; form net-^ works. Networks broken up by l y sozyme but c e l l s not l y s e d  ? negative  1.698 g/cm  e) s h i n y , y e l l o w , f l a t colony w i t h i r r e g u l a r margin; h y d r o l y zes agar  non-motile; l o n g f i l a m e n t o u s rods  positive  f)  non-motile rod ocnegative curring singly and i n s h o r t c h a i n s  1.715 g/cm"  g) d i f f u s e w h i t e , f l a t colony with i r r e g u l a r margin  non-motile rod o c curring singly, p a i r e d and i n short chains  1.716 g/cm  h) s h i n y , orange, f l a t colony with e n t i r e margin  nonnmojfcilei?., l o n g positive r o d s , some i n c h a i n s curved ( f i l a m e n t s ? )  s h i n y , creamy-white domed c o l o n y w i t h i r r e g u l a r margin  1.743 g/cm"  long,  1.681 g/cm (polysaccharide?) 3  negative  1.697 g/cm  3  1.706 g/cm"  25 The one  degree o f c o n t a m i n a t i o n i n c h l o r o p l a s t p r e p a r a t i o n s  b a c t e r i u m p e r 6500 c h l o r o p l a s t s t o one b a c t e r i u m p e r 107,000  plasts.  I f a c h l o r o p l a s t contains  the b a c t e r i a may have c o n t r i b u t e d preparation. DNA  ranged from chloro-  the same amount o f DNA as a b a c t e r i u m , 0.001% to 0.015% o f the DNA i n the f i n a l  Assuming t h a t a c h l o r o p l a s t c o n t a i n s  one t e n t h  the amount o f  i n a b a c t e r i u m , a t most the amount o f b a c t e r i a l DNA i n the f i n a l  paration  pre-  c o u l d be 0.15%.  E l e c t r o n Microscopy Sections  of i s o l a t e d c h l o r o p l a s t s observed w i t h the e l e c t r o n m i c r o -  scope showed m o s t l y i n t a c t bulging  o f the membranes  osmotic changes d u r i n g  c h l o r o p l a s t s , a l t h o u g h i n many cases t h e r e  ( f i g . 1).  This bulging  was  may have been caused by  the f i x a t i o n o f the c h l o r o p l a s t s .  No b a c t e r i a l p r o -  f i l e s were seen. For  c h l o r o p l a s t s which were sedimented through a b u f f e r e d  l u t i o n , one m i t o c h o n d r i a l  sucrose so-  p r o f i l e was seen f o r every 5 c h l o r o p l a s t  profiles.  Fewer m i t o c h o n d r i a were observed when the c h l o r o p l a s t s were sedimented through A medium d u r i n g  the i s o l a t i o n p r o c e d u r e .  In t h i s case, one m i t o -  c h o n d r i a l p r o f i l e p e r t e n c h l o r o p l a s t p r o f i l e s was seen. except f o r a few p r e l i m i n a r y sucrose s o l u t i o n during  preparations,  the f i n a l  A medium was used r a t h e r  s t e p s o f the c h l o r o p l a s t  Although o n l y one m i t o c h o n d r i a l  For t h i s  reason, than  isolation.  p r o f i l e was seen p e r 10 c h l o r o p l a s t  p r o f i l e s , the a c t u a l r a t i o o f m i t o c h o n d r i a t o c h l o r o p l a s t s was p r o b a b l y much h i g h e r than t h i s .  Since a c h l o r o p l a s t i s l a r g e r than a m i t o c h o n d r i o n ,  the p r o b a b i l i t y o f s e c t i o n i n g through a c h l o r o p l a s t i s g r e a t e r p r o b a b i l i t y o f s e c t i o n i n g through a m i t o c h o n d r i o n .  than t h e  The mean volume of an  26  F i g u r e 1:  T h i n s e c t i o n s of AcetcLbulavia fixed i n glutaraldehyde  medttevranea  and p o s t - f i x e d OsO^.  were s t a i n e d w i t h u r a n y l a c e t a t e Bar = 1 y(x 10,000.)  chloroplasts Sections  f o l l o w e d by l e a d  citrate.  27  Acetabulavia  c h l o r o p l a s t i s 8 ,p  3  (55) .  a m i t o c h o n d r i o n , and by measuring  Assuming an e l l i p s o i d shape f o r  the diameters o f m i t o c h o n d r i a l p r o f i l e s  Acetabulavia  observed w i t h the e l e c t r o n microscope, the mean volume o f an 3 m i t o c h o n d r i o n i s c a l c u l a t e d to be a p p r o x i m a t e l y 0.4  u .  Assuming t h a t  an  e q u a l number o f c h l o r o p l a s t s and m i t o c h o n d r i a are randomly d i s t r i b u t e d i n a g i v e n volume of embedding medium ( t h i s may then the chances  not be a v a l i d  assumption),  o f s e c t i o n i n g through a c h l o r o p l a s t are 20 times g r e a t e r  than the changes of s e c t i o n i n g through a m i t o c h o n d r i o n .  Thus, the a c t u a l  r a t i o c o u l d be as h i g h as two m i t o c h o n d r i a ^ f o r e v e r y c h l o r o p l a s t . The amount of DNA it  appears  i n an  Acetabulavia  mitochondrion i s not known, but  to be l e s s than i s i n a c h l o r o p l a s t  s t u d i e s of the c y t o p l a s m i c DNAs of 10 times more c h l o r o p l a s t DNA  (75).  Acetabulavia,  From buoyant  t h e r e appears  than m i t o c h o n d r i a l DNA  (32).  t h a t a m i t o c h o n d r i o n c o n t a i n s one t e n t h the amount o f DNA c h l o r o p l a s t , m i t o c h o n d r i a l DNA i n the f i n a l p r e p a r a t i o n . i s no measurable (fig.  2 ) , p a 1.714  g/cm  3  Buoyant d e n s i t y and Tm o f  Acetabulavia  to be  about  Assuming, then, that i s i n a  c o u l d c o n t r i b u t e as much as 20% of the  In DNA  amount of DNA  density  DNA  from p u r i f i e d c h l o r o p l a s t s , however, t h e r e  a t the buoyant  d e n s i t y o f m i t o c h o n d r i a l DNA  (32).  Acetabulavia  c h l o r o p l a s t DNA  chloroplast  DNA  from two d i f f e r e n t p r e p a r a t i o n s was  t r i f u g e d i n an a n a l y t i c a l C s C l d e n s i t y g r a d i e n t .  cen-  In b o t h c a s e s , a s i n g l e  3 peak w i t h a buoyant  d e n s i t y o f 1.702  different  from the buoyant  taminants  (table I ) .  (51), p = 1.702  g/cm  3  g/cm  was  d e n s i t y of the DNA  observed  ( f i g . 2).  This i s  o f any of the b a c t e r i a l  con-  U s i n g the r e l a t i o n s h i p g i v e n by S c h i l d k r a u t e t a l . corresponds t o a base c o m p o s i t i o n o f 42.8%  G+C.  A  28  F i g u r e 2:  Densitometer t r a c i n g s o f u l t r a v i o l e t photographs o f  Aeetabulavia  meditevvanea  l y t i c a l density-gradient  c h l o r o p l a s t DNA  a f t e r ana-  centrifugation i n CsCl. 3  Micrococcus  lysodeikticus  used as d e n s i t y  DNA (p = 1.731 g/cm ) was  reference.  29 buoyant d e n s i t y o f 1.702 g/cm in  3  p r e d i c t s a Tm o f 87.0°C i n SSC and 71.2°  0.1 x SSC, u s i n g the f o l l o w i n g e q u a t i o n s  g i v e n by Mandel e± al_ (41):  Tm (SSC) = 418.2 (p- 1.494) Tm (SSC/10) = 512.2 (p-1.563) In one attempt to determine t h e Tm i n SSC, the curve began t o l e v e l a f t e r a hyperchromic i n c r e a s e o f about 31%, b u t t h i s was f o l l o w e d by a sudden hyperchromic r i s e above 95° ( f i g . 3 ) . T h i s sudden i n c r e a s e i n hyp e r c h r o m i c i t y may have been due t o e v a p o r a t i o n o f the s o l v e n t , s i n c e about 1.5 ml o f sample was c o n t a i n e d i n a c u v e t t e which has a c a p a c i t y o f 1.8 m l . Another p o s s i b l e e x p l a n a t i o n i s t h a t t h i s sudden i n c r e a s e was an a r t i f a c t produced by a gas bubble.  T a k i n g 31.5% as the maximum h y p e r c h r o m i c i t y ,  the Tm i n SSC o f the Aeetabulavia = 90.5  for  E. coli  c h l o r o p l a s t DNA i s 86.7°, based on a Tm  K 12 DNA.(41).  T h i s i s i n good agreement w i t h the p r e -  d i c t e d v a l u e o f 87.0°.  Aeetabulavia  c h l o r o p l a s t DNA had 22.8% h y p e r c h r o m i c i t y i n 0.1 x SSC,  w i t h a Tm o f 70.7° ( f i g . T h i s i s 0.6° lower  3) based on a Tm = 75.7° f o r  E. coli  than p r e d i c t e d , but t h i s may be due t o the low m o l e c u l a r  weight o f the DNA (20,21).  The m e l t i n g t r a n s i t i o n was n o t smooth.  a l s o , i s p o s s i b l y due t o the f a c t t h a t t h i s DNA was sheared c u l a r weight  (about  K 12 DNA (41).  7.5 x 10^ d a l t o n s ) .  molecular heterogeneity i n  Aeetabulavia  t o a low mole-  I f t h e r e i s a h i g h degree o f i n t r a c h l o r o p l a s t DNA, then s h e a r i n g o f  the DNA c o u l d produce fragments o f v a r y i n g base c o m p o s i t i o n s . p l o t o f the thermal ple (23).  transition of  This,  Aeetabulavia  A differential  c h l o r o p l a s t DNA shows m u l t i -  peaks ( f i g . 4 ) , which i s a l s o i n d i c a t i v e o f i n t r a m o l e c u l a r h e t e r o g e n e i t y  30  F i g u r e 3:  Melting  curves o f n a t i v e DNA.  meditewanea  c h l o r o p l a s t DNA  Acetabulavia  meditewanea  (A—A)  E. coli  DNA  (•••)  Acetabulavia  melted i n 0.1 x SSC;  c h l o r o p l a s t DNA  melted i n SSC.  (ooo)  melted i n SSC;  30a  Ui  1-3  in 1-2 CM  1-1  VO 30  40  50  60  70  TEMPERATURE (°C)  80  90  31  Figure  4:  D i f f e r e n t i a l m e l t i n g p l o t of n a t i v e DNA (o—o)  Aoetabularia  (•=-•) E. coll  meditewanea DNA.  i n 0.1  chloroplast  x DNA;  SSC  31a  32  Acetabulavia for  melting.  c h l o r o p l a s t DNA The  perchromicity)  t r a n s i t i o n w i d t h (°C between 17%  of melting  c o u l d be p a r t l y due may  a l s o be  shows a v e r y broad t r a n s i t i o n w i d t h  i n SSC  i s 8.2°  to the low m o l e c u l a r  and  and  i n 0.1  83%  x SSC  of t o t a l  hy-  i s 9.7°.  This  weight of the DNA  (20,21), but  i n d i c a t i v e o f a h i g h degree of i n t r a m o l e c u l a r  heterogeneity  (42).  Renaturation The  and k i n e t i c complexity  of  Acetabulavia  r a t e p l o t s f o r the r e n a t u r a t i o n of  are g i v e n i n f i g u r e 5.  There appears to be  chloroplast  Acetabulavia  DNA  chloroplast  DNA  a s m a l l amount of a f a s t  re-  n a t u r i n g component, r e p r e s e n t i n g about 3% of the t o t a l DNA.  This i s pro-  bably  t h e r e was  due  to m i t o c h o n d r i a l DNA.  considerable l a g before p l a i n , but may t i o n i n 0.1  be  x SSC w i t h  i n t h i s sample was  of the  the n e u t r a l i z e d sample was g r a d i e n t , no band was It  (20x)  SSC  v e r y low  solution. (about  Aeeta&Uikavia subsequently  detected,  Acetabulavia  the c h l o r o p l a s t DNA  and  to  Also,  I t i s probable  ex-  soluthe  6 yg/ml), and  was  c h l o r o p l a s t DNA  alkali  this  was  denatured.  observed.  When  run i n an a n a l y t i c a l C s C l d e n s i t y  suggesting  t b a t the DNA  c h l o r o p l a s t DNA,  had been degraded.  to the presence o f  s i n c e the m e l t i n g b e h a v i o r  i t s buoyant d e n s i t y i n C s C l c o r r e l a t e w e l l  t h a t f r e e r a d i c a l s are generated d u r i n g the  unof  (41,42).  problem o f a l k a l i l a b i l i t y has been encountered w i t h m i t o c h o n d r i a l  (10).  a  for this lag.  i s v e r y u n l i k e l y t h a t t h i s a l k a l i l a b i l i t y i s due  u s u a l bases i n  The  This i s d i f f i c u l t  r e n a t u r a t i o n experiment, the DNA  No h y p e r c h r o m i c i t y  ( f i g . 5b)  to i n s u f f i c i e n t m i x i n g o f the DNA  the c o n c e n t r a t e d  be p a r t of the reason In the f i r s t  experiment  the r e a c t i o n proceeded.  an a r t i f a c t due  c o n c e n t r a t i o n of DNA may  In one  DNA  DNA  33  F i g u r e 5:  Renaturation rate p l o t s f o r chloroplast  Acetabulavia  meditewanea  DNA.  (a) Experiment 2:  DNA  r e n a t u r e d i n SSC (b) Experiment 3:  c o n c e n t r a t i o n approximately  at 63 + 0.5°; k^ = 5.90  DNA  (c) Experiment 4: DNA  1 mole ''"sec ^  c o n c e n t r a t i o n approximately  r e n a t u r e d i n 2 x SSC  a t 65 + 0.5°; k^ = 5.00  concentration approximately  r e n a t u r e d i n 2 x SSC  at 65 + 0.5  ; k  2  15 yg/ml;  = 7.62  6 yg/ml;  1 mole ''"sec 11 yg/ml; 1 mole  sec  k^ v a l u e s c o r r e c t e d f o r i o n i c s t r e n g t h of s o l v e n t and normal i z e d to 50%  G+C  33a  a)  ^  1-0jr  1  0  1  1  1000 2000  1  1  1  1  r  — • • — i  3000 4000 5000 (50min) TIME(sec)  1  1 —  6000 7000 dOOmin)  34  i s o l a t i o n procedure  (10) and  these a t t a c k the N - g l y c o s i d i c bond  T h i s r e s u l t s i n base e l i m i n a t i o n and (49).  (49).  subsequent c h a i n breakage a t pH > 7  T h e r e f o r e , i n subsequent experiments,  heat d e n a t u r a t i o n was  used  r a t h e r than a l k a l i n e d e n a t u r a t i o n . In  col-i  experiment  DNA  4, a f t e r the DNA  88% r e n a t u r e d ;  had been r e n a t u r e d f o r 34 hours  Aoetahularia  c h l o r o p l a s t DNA  q u i c k l y c o o l e d and d i a l y s e d i n t o 0.1 the DNA  was  melted.  There was  x SSC  59%  i n the c o l d .  some l o s s of O.D.  p r o b a b l e t h a t some d e g r a d a t i o n of the DNA  t i o n r e a c t i o n and subsequently  renatured  lower  AoetabulcLvta  than the Tm  DNA  to d i a l y s i s .  It  o c c u r r e d d u r i n g the r e n a t u r a -  samples are shown i n f i g u r e  c h l o r o p l a s t DNA  has  of n a t i v e c h l o r o p l a s t DNA.  has a Tm which i s lowered by 0.7°..  E. ooli  T h i s l o w e r i n g of the Tm  DNA  0.7°  also  i n d i c a t e s about  (40).  renaturation rate constants, a l k a l i n e sedimentation  and k i n e t i c c o m p l e x i t i e s of a number o f DNA  6.  a Tm o f 70.0°, which i s Renatured  1% mismatched base p a i r s i n the r e n a t u r e d DNA The  Following t h i s ,  s m a l l fragments were l o s t d u r i n g d i a l y s i s .  The m e l t i n g curves f o r the r e n a t u r e d DNA The  i t was  f o r the c h l o r o p l a s t  sample, even a f t e r c o r r e c t i n g f o r the volume change due is  renatured)  (E.  coefficients  samples a r e p r e s e n t e d i n t a b l e  9 II.  The k i n e t i c c o m p l e x i t y of  E. coli  DNA  agreement w i t h i t s a n a l y t i c a l complexity of  these experiments,  should be 2.5  (74).  x 10  daltons, i n  The v a l u e s o b t a i n e d i n some  however, are lower than t h i s .  The most p r o b a b l y  ex-  p l a n a t i o n i s t h a t the v a l u e s f o r the a l k a l i n e s e d i m e n t a t i o n c o e f f i c i e n t s in  error.  used  Sedimentation  c o e f f i c i e n t s were determined  f o r the r e n a t u r a t i o n r e a c t i o n .  temperature  a f t e r the DNA  A f t e r t h i s prolonged  had  are been  exposure to a h i g h  t h e r e would have been some d e g r a d a t i o n of the DNA  (22) , r e s u l t i n g  35  F i g u r e 6:  Thermal t r a n s i t i o n p r o f i l e s o f n a t i v e and r e n a t u r e d melted i n 0.1  x SSC.  c h l o r o p l a s t DNA; r e n a t u r e d DNA, n a t i v e DNA,  (a)  (•••)  n a t i v e DNA,  Tm = 70.0°.  Tm = 75.7°;  Acetabulavia  (b)  DNA  meditewanea Tm = 70.7°;  E. coli  DNA;  (ooo) r e n a t u r e d DNA,  (ooo) (•••)  Tm = 75.0°.  1-3,  5a  1-2 h  o oo  in CNJ  1-1r-  o o  ~ o ooo' O 0 oo  o  o  1-Oh  •  30  •  mo  o •  •  a  40 50 60 70 T E M P E R A T U R E (°C)  80  30  40 50 60 70 T E M P E R A T U R E (°C)  80  Table I I .  Exp. No.  K i n e t i c complexities  o f DNA from d i f f e r e n t sources  Source o f DNA  3  -1 ,-1 (mole 1 ) (1 mole sec sec 1)  1  b a c t e r i o p h a g e T4  3.01x10  1  E.  3.57x10  coli  ,pH13 20,w meas. after  -5 -5  meas before  calculated  197  24  1.5 x 10  11.60  14.9  9 1.4 x 10 2.5 x 10'  24  E.  coli  -5  5.73x10  13.57  13  1.0 x 10< 2.6 x 10'  28  33  E. E.  coli  -5  3.92x10  coli  §5  4.54x10  5  E.  coli  6.00x10  2  Aeetabulavia meditevvanea  4.39x10  -5 -5  K i n e t i c Complexity (daltons)  13  1.0 x 10^ 2.5 x 10'  15.7  1.4 x 10j 2.5 x 10'  5.31  6.88  6.05  10  13.4  2.3 x 10'  13  8 8.7 x 10 2.3 x 10'  19.5  1.7 x 10^ 4.5 x 10'  11  8 8.1 x 10 1.4 x \V  5.90  chloroplasts  Aeetabulavia  1.82x10  -5  5.00  chloroplasts  Aeetabulavia  3.10x10  -5  7.62  chloroplasts  Co  * pH13 S„„ 20,w  corrected  to o b t a i n  a k i n e t i c complexity o f 2.5 x 10  9  ON  daltons  for  E. coli  DNA  37  in  lower v a l u e s f o r the s e d i m e n t a t i o n c o e f f i c i e n t s .  with  E. coli  I n one  experiment  DNA, an a l i q u o t o f DNA f o r d e t e r m i n a t i o n o f the s e d i m e n t a t i o n  c o e f f i c i e n t was taken a t the s t a r t o f t h e experiment,  immediately  after  9 In t h i s case, a value o f 2 . 3 x 1 0  the DNA had been heat denatured.  d a l t o n s was o b t a i n e d f o r the k i n e t i c c o m p l e x i t y  ( t a b l e I I , experiment  5).  In  a value o f  was  another  experiment  ( t a b l e I I , experiment  2),  S^Q"^  3  =  13  determined  on a sample o f DNA a f t e r i t had been used i n the r e n a t u r a t i o n  reaction.  With another  sample o f sheared DNA from the same b a t c h , but  which had n o t been heat denatured  and r e n a t u r e d ,  S?^F"  3  =  Using  28.  13  as  20,w  9  the a l k a l i n e s e d i m e n t a t i o n c o e f f i c i e n t , a k i n e t i c c o m p l e x i t y o f 1 . 0 x 1 0 d a l t o n s i s c a l c u l a t e d f o r E. aoli DNA, b u t u s i n g S^?"*" = 2 8 , the k i n e t i c 2 0 ,w 3  9 complexity o f  E. aoli  DNA i s  2.6  x  10  daltons.  T h e r e f o r e , the v a l u e s o f  the s e d i m e n t a t i o n c o e f f i c i e n t s were c o r r e c t e d t o o b t a i n a k i n e t i c ty  of  2.5  x  10  daltons f o r  E. coli  DNA.  A c o r r e s p o n d i n g c o r r e c t i o n was  made f o r the s e d i m e n t a t i o n c o e f f i c i e n t s o f t h e DNA.  Acetabulavia  chloroplast  T h i s c o r r e c t i o n assumes t h a t the c h l o r o p l a s t DNA was degraded to the  same degree as t h e  E. coli  DNA.  T h i s may not be a v a l i d  assumption.  a l o o k a t the c o r r e c t e d v a l u e s g i v e n i n t a b l e I I would suggest assumption i s n o t v a l i d .  One would expect  p e r i o d o f time, y e t i t has the s m a l l e s t c o r r e c t i o n .  the c o r r e c t i o n a p p l i e d i n experiment was made t o determine  Indeed,  that this  the DNA i n experiment  the most degraded, s i n c e i t was exposed to a h i g h temperature est  complexi-  4 t o be  f o r the long-  On the o t h e r hand,  3 i s p r o b a b l y too l a r g e .  a c o r r e c t i o n f a c t o r f o r the a l k a l i n e  An  attempt  sedimentation  c o e f f i c i e n t s by s h e a r i n g b a c t e r i a l DNA under the same c o n d i t i o n s as had been used t o shear the c h l o r o p l a s t DNA (see below).  38 The k i n e t i c complexity o f  Acetabulavia  meditewanea  chloroplast  DNA  g is  a t l e a s t 8.1 x 10  d a l t o n s , and i s p r o b a b l y g r e a t e r than t h i s .  The  average r e s u l t o f t h r e e s e p a r a t e experiments g i v e s a k i n e t i c c o m p l e x i t y 9 of  1.1 + 0.4 x 10  daltons.  T h i s agrees c l o s e l y w i t h an a n a l y t i c a l com-  9 -15 p l e x i t y o f 1.4 x 10 d a l t o n s (2.3 x 10 g) o f DNA as determined by Green (Unpublished).  Thus, i t appears p r o b a b l e t h a t the c h l o r o p l a s t DNA  as one unique sequence, w i t h no r e p e a t e d segments. be two c o p i e s o f the unique  exists  At most, t h e r e c o u l d  sequence.  Sedimentation c o e f f i c i e n t s If  the method o f s h e a r i n g produced  the same s i z e o f DNA  fragments  f a i r l y r e p r o d u c i b l y , then any DNA sample, c o u l d be taken and sheared the  same c o n d i t i o n s as were used f o r s h e a r i n g the DNA i n the r e n a t u r a t i o n  experiments.  The a l k a l i n e s e d i m e n t a t i o n c o e f f i c i e n t s c o u l d then be d e t e r -  mined f o r these samples ing  under  and the v a l u e s o b t a i n e d c o u l d be used i n c a l c u l a t -  the k i n e t i c c o m p l e x i t i e s .  T h i s was done, u s i n g p u r i f i e d DNA, which was  e x t r a c t e d from one o f the b a c t e r i a l contaminants.  S o l u t i o n s o f the DNA  were a d j u s t e d t o the c o n c e n t r a t i o n s used i n the r e n a t u r a t i o n  experiments.  Three a l i q u o t s o f each c o n c e n t r a t i o n o f DNA were taken and each sheared once o r twice w i t h a 27 gauge n e e d l e , depending used p r e v i o u s l y .  aliquot  on the c o n d i t i o n s  The DNA samples were c o n c e n t r a t e d w i t h Aquacide I I , d i a -  l y z e d i n t o 0.1 x SSC, and then the a V l k a l i n e s e d i m e n t a t i o n c o e f f i c i e n t s were determined.  The r e s u l t s o f these experiments are p r e s e n t e d i n t a b l e  III. It  can be seen t h a t w i t h i n the c o n c e n t r a t i o n range used, the concen-  t r a t i o n o f DNA had l i t t l e  e f f e c t on the s i z e o f the DNA fragments  produced  39  Table I I I . Alkaline  sedimentation c o e f f i c i e n t s o f sheared Samples  DNA  DNA c o n c e n t r a t i o n ( y g / m l )  sheared: q b  pH13 20,w  mean:  6.5  8  10  12  15  x2  xl  x2  xl  xl  25.6  25.8  26.2  26.6  25.4  23.8  24.2  25.3  26.4  27.2  25.0  31.7  26.6  29.2  25.5  27 . 4 + 1 . 5 6  26.0+1.01  27.2+3.92 • 26 +0.21  28.7+1.12  b y s h e a r i n g w i t h a 27 gauge n e e d l e . mentation  coefficients obtained  T h e r e w a s some v a r i a t i o n i n t h e s e d i -  f o r d i f f e r e n t DNA s a m p l e s a t t h e s a m e  c e n t r a t i o n , p a r t i c u l a r l y w h e n t h e DNA w a s s h e a r e d e n c e s i n S??^"  3  only once.  These  con-  differ-  f o r d i f f e r e n t s a m p l e s o f DNA c a n l e a d t o c o n s i d e r a b l e  20,w variation i n the calculated value o f the kinetic complexity, table IV using the k  2  determined  f o r E. ooli  DNA i n e x p e r i m e n t  each case, the value f o r the k i n e t i c complexity t a t i o n c o e f f i c i e n t s determined  i s too high.  i n these experiments  as shown i n one.  In  The sedimen-  are generally  higher  t h a n t h e v a l u e s m e a s u r e d f o r t h e DNA s a m p l e s u s e d i n t h e r e n a t u r a t i o n e x periments.  This gives support  t o t h e i d e a t h a t s o m e DNA d e g r a d a t i o n o c -  curred during the renaturation experiments. E. ooli  On t h e o t h e r h a n d , b o t h t h e  DNA a n d t h e c h l o r o p l a s t DNA ( p a r t i c u l a r l y t h e c h l o r o p l a s t DNA)  f o r t h e r e n a t u r a t i o n e x p e r i m e n t s wer:e h a n d l e d  more e x t e n s i v e l y than  this  used  40  T a b l e IV.  V a r i a t i o n i n k i n e t i c complexity with  different  v a l u e s o f S^,20 ,w Source of DNA  DNA concentration 12 yg/ml  12 yg/ml  ^20^w '  k i n e t i c complexity (daltons)  11.6 1 mole sec~l  14.9 24.0  1.4 2.5  X X  10 109  11.6 1 mole sec~l  26.4^  2.8  X  10  9  27.4^  2.9  X  10  9  29.2^  3.2  X  10  9  9  * d a t a taken from experiment  1.  taken from t a b l e I I I .  b a c t e r i a l DNA and, t h e r e f o r e , p o s s i b l y had a lower m o l e c u l a r weight bef o r e s h e a r i n g w i t h the n e e d l e .  S i n c e the s e d i m e n t a t i o n  determined  give k i n e t i c complexities f o r  i n these experiments  coefficients  E. coli  which are too l a r g e , i t was n o t p o s s i b l e to use a c o r r e c t i o n f a c t o r on  them.  DNA based  41  DISCUSSION  Aeetabulavia  meditevvanea  c h l o r o p l a s t DNA  has a buoyant d e n s i t y  3 in  C s C l o f 1.702  g/cm  .  T h i s agrees  q u i t e c l o s e l y to the v a l u e of  3 1.704 and  g/cm  r e p o r t e d by Green e t a l . (32) and H e i l p o r n and Limbosch  i n d i c a t e s a basedcomposition  c h l o r o p l a s t DNA  i n SSC  base c o m p o s i t i o n ,  i s 86.7°.  G+C.  T h i s g i v e s a v a l u e of 43% G+C  as the s t a n d a r d .  The  conclusion.  (36, 41, 42).  Aeetabulavia  Aeetabulavia  meditevva-  Chromatographic data o f G i b o r  c h l o r o p l a s t DNA  which was  then chromatographed showed o n l y deoxyadenosine,  deoxycytosine  and  Aeetabulavia 0.1  x SSC.  content.  p l a s t DNA of  the DNA  T h i s i s 0.6°  c h l o r o p l a s t DNA lower  T h i s l o w e r i n g of the Tm (20,20)).  i s v e r y broad.  thymidine,  than expected  melts w i t h a Tm f o r a DNA  i s p r o b a b l y due  T h i s i s p a r t l y due  o f 43%  to the low  The m e l t i n g t r a n s i t i o n o f  of 70.7° G+C  molecular  Aeetabulavia  chloro-  to the low m o l e c u l a r weight  (20,21) but c o u l d a l s o be i n d i c a t i v e of a h i g h degree o f  molecular heterogeneity  (42).  The  observed  intra-  unevenness of the m e l t i n g  t r a n s i t i o n , e s p e c i a l l y n o t i c e a b l e when the DNA may  enzy-  deoxyguanosine (26).  meditevvanea  weight of the DNA  supports  hydrolyzed  m a t i c a l l y and  in  (41)  c a l c u l a t e d from the buoyant d e n s i t y and the m e l t i n g tempera-  c h l o r o p l a s t DNA  this  f o r the  c l o s e agreement of the base  t u r e i n d i c a t e s the absence of any unusual bases i n nea  Aeetabulavia  The Tm o f  as c a l c u l a t e d from e q u a t i o n 5 o f Mandel et_ al_.  u s i n g _E. c o l i K 12 DNA compositions  of 42.8%  (34),  was  melted  i n 0.1  x  SSC,  a l s o be i n d i c a t i v e of a h i g h degree o f i n t r a m o l e c u l a r h e t e r o g e n e i t y .  T h i s uneven m e l t i n g t r a n s i t i o n i s s i m i l a r to t h a t r e p o r t e d f o r the c h o n d r i a l DNA  of  Dvosophila  melanogastev  (47).  P o l a n e t a l . (47)  mitoattribute  42 the m u l t i t r a n s i t i o n a l b e h a v i o r of  D. melanogastev  m i t o c h o n d r i a l DNA  to  d i f f e r e n c e s i n base composition i n v a r i o u s p o r t i o n s of the m o l e c u l e . d i f f e r e n t i a l p l o t of the m e l t i n g t r a n s i t i o n o f DNA  A. meditewanea  A  chloroplast  shows m u l t i p l e peaks, which supports the c o n c l u s i o n t h a t t h i s DNA  a h i g h degree o f i n t r a m o l e c u l a r h e t e r o g e n e i t y . c h l o r o p l a s t DNAs from (5,72) and  Euglena  Chlovella  gracilis  pyrenoidosa  has  The m e l t i n g c u r v e s o f  (6),  Chlamydomonas  reinhavdtii  (65) show c o n s i d e r a b l e i n t r a m o l e c u l a r h e t e r o -  g e n e i t y i n these DNAs as w e l l . The k i n e t i c c o m p l e x i t y o f  Acetabulavia  c h l o r o p l a s t DNA,  based  on  the  9 average  v a l u e o b t a i n e d i n t h r e e s e p a r a t e experiments  g  d a l t o n s , w i t h the l e a s t v a l u e b e i n g 8.1  E. coli  i s 1.1 + 0.4  DNA  which was  x 10  x  10  d a l t o n s (see t a b l e I I ) .  r e n a t u r e d a t the same time as the c h l o r o p l a s t  under the same c o n d i t i o n s , has  a k i n e t i c complexity  lower than the  DNA, litera-  9 t u r e v a l u e o f 2.5  x 10  d a l t o n s (74).  T h i s i s p r o b a b l y due  the v a l u e s of the a l k a l i n e s e d i m e n t a t i o n c o e f f i c i e n t s T h e r e f o r e , the k i n e t i c c o m p l e x i t y of  Acetabulavia  to e r r o r s i n  (see R e s u l t s ) .  meditewanea  chloroplast  9 DNA  i s p r o b a b l y g r e a t e r than 1.1  the amount o f DNA  x 10  daltons.  T h i s would i n d i c a t e t h a t —16 7  p e r c h l o r o p l a s t i s more than the 1 x 10  g (6x10  dal-  tons) e s t i m a t e d by Gibor and Izawa (27). I f the a n a l y t i c a l complexity o f 9 _15 1.4 x 10 d a l t o n s (2.3 x 10 g) determined by Green (unpublished) i s c o r r e c t , then a t most t h e r e c o u l d be two c o p i e s o f the DNA n u c l e o t i d e s e -  g  quence, based of  the DNA  on a k i n e t i c complexity o f 8.1  x 10  daltons.  If a  i s h i g h l y r e p e t i t i o u s , t h i s would have been e v i d e n t as a s e p a r -  ate k i n e t i c component i n the r e n a t u r a t i o n r e a c t i o n (12, 13, 14, v e r y s m a l l amount ( a p p r o x i m a t e l y 3% o f the t o t a l DNA) ing  fraction  component was  observed but t h i s i s most l i k e l y due  74).  A  of a f a s t e r renaturto mitochondrial  43  DNA.  S i n c e 8.1  x 10  g  d a l t o n s r e p r e s e n t s a minimum v a l u e , and  the average  9 v a l u e f o r the k i n e t i c complexity too low due it  (1.1 + 0.4  x 10  daltons) i s probably  to e r r o r s i n the v a l u e s o f the s e d i m e n t a t i o n  coefficients,  seems l i k e l y t h a t there i s no n u c l e o t i d e sequence r e p e t i t i o n i n  Aeetabulavia  meditevvanea  c h l o r o p l a s t DNA.  On  the o t h e r hand, s i n c e  2.3  x 10 "'"^g r e p r e s e n t s an average amount of DNA  per c h l o r o p l a s t , i t i s p o s s i -  ble  t h a t w h i l e some c h l o r o p l a s t s c o n t a i n one  copy o f the genome, o t h e r s  may  c o n t a i n two  if,  d u r i n g some c h l o r o p l a s t d i v i s i o n s , a l l of the DNA  o r more c o p i e s .  Other c h l o r o p l a s t s may  c h l o r o p l a s t and none to the o t h e r .  not c o n t a i n  goes to one  T h i s would be compatible  s e r v a t i o n s o f Woodcock and Bogorad (75) t h a t no DNA  daughter  w i t h the  the amount present was  t h a t i n those  variable.  The k i n e t i c c o m p l e x i t i e s of c h l o r o p l a s t DNAs from a number of are shown i n t a b l e V. c h l o r o p l a s t DNA  The k i n e t i c c o m p l e x i t y  of  Aeetabulavia  component of k i d n e y bean c h l o r o p l a s t DNA the k i n e t i c c o m p l e x i t y  of  Aeetabulavia  p u r i t y o f the DNA  of  t h e r e i s some q u e s t i o n as to the C h i l t o n , p e r s o n a l communica-  T h i s throws doubt on the v a l i d i t y of h i s h i g h v a l u e  Aeetabulavia  u a l l y l a r g e — b u t then,  Aeetabulavia  larger kinetic  Crandall's results  the k i n e t i c c o m p l e x i t y o f k i d n e y bean c h l o r o p l a s t DNA.  n e t i c complexity  meditevvanea  i s the o n l y v a l u e t h a t approaches  used i n h i s experiments (M.D.  Green).  The  c h l o r o p l a s t DNA.  have never been p u b l i s h e d , however, and  for  sources  i s l a r g e r than the c o m p l e x i t i e s of o t h e r c h l o r o p l a s t DNAs  which have been i n v e s t i g a t e d by r e n a t u r a t i o n k i n e t i c s .  t i o n to B.R.  ob-  c o u l d be d e t e c t e d i n  65-80% of c h l o r o p l a s t s , u s i n g f o u r d i f f e r e n t methods; and c h l o r o p l a s t s w i t h d e t e c t a b l e DNA  DNA  Aeetabulavia  meditevvanea  Thus, the k i -  c h l o r o p l a s t DNA  i s an unusual organism.  seems unusThe  f a c t that  can s u r v i v e f o r l o n g p e r i o d s f o l l o w i n g e n u c l e a t i o n makes  one  44  Table V:  K i n e t i c c o m p l e x i t i e s o f c h l o r o p l a s t DNAs from v a r i o u s sources C h l o r o p l a s t DNA k i n e t i c comAnalytical plexity complexity (daltons) (daltons)  Organism  Aeetabulavia meditevvanea  1.1 + 0.4 x 10'  Euglena  gvaoilis  1.8 x 10  Ch lanry domonas veinhavdtii  1.94 x 10  1.4 x 10'  8  2 x 10  S t u t z (64)  8  1-10 x 10  5.16 x 10'  6  8  Chlovella pyvenoidosa  3 x 10 1.3 x 10  E  Phaseolus vulgaris  7.4 x 10  £  Reference  B a s t i a et^ a l . (5) W e l l s and Sager (72) Bayen and Rode (6)  7.2 x 10  8  C r a n d a l l (19)  7.5 x 10*  (kidney bean) Lettuce  3 x 10  2 x 10'  6  1.2 x 10  8  Pea  9.5 x 10  Tobacco  1.14 x 10  W e l l s and B i r n s t i e l (71) Kolodner and Tewari (38)  8  Tewari and Wildman (67)  Suspect t h a t t h i s a b i l i t y may be r e l a t e d to the amount o f g e n e t i c t i o n i n the c h l o r o p l a s t s .  informa-  I t h a r d l y seems s u r p r i s i n g t h a t the c h l o r o p l a s t  DNA o f a u n i n u c l e a t e c e l l as l a r g e as  Aeetabulavia  has a g r e a t e r unique  sequence l e n g t h than the few c h l o r o p l a s t DNAs o f o t h e r organisms which have been s t u d i e d to date.  45 Proponents of the  symbiotic  suggest t h a t t h e r e was in  the  theory of the o r i g i n of  a reduction  i n the amount o f g e n e t i c  c h l o r o p l a s t as t h i s i n f o r m a t i o n  symbiont to the n u c l e a r  genome d u r i n g  p l a s t became an i n t e g r a l p a r t o f the  Acetabulavia  then perhaps the  cell  to the  been s t u d i e d . studied  chloroplasts  was the  cell  information  t r a n s f e r r e d from the course of t i m e , and (61).  organelle-  the  I f t h i s i s the  chloro-  case,  c h l o r o p l a s t s have not become i n t e g r a t e d  same degree as c h l o r o p l a s t s of o t h e r p l a n t s Bayan and  into  t h a t have  Rode (6) suggest t h a t s i n c e most c h l o r o p l a s t DNAs  are q u i t e s i m i l a r i n terms o f k i n e t i c c o m p l e x i t y and  heterogeneity,  t h i s i s din agreement w i t h the t h e o r y that c h l o r o p l a s t s o f d i f f e r e n t organisms might have had  a common o r i g i n .  I t i s p o s s i b l e , however, t h a t  p l a s t s arose more than once, r a t h e r than as a r e s u l t o f a s i n g l e ary  evolution-  event. Two  k i n e t i c components have been observed f o r the  from some sources (6, 19, c h l o r o p l a s t DNA ing  chloro-  there  component was  by m i t o c h o n d r i a l  veinhavdtii  71,  i s o n l y one  observed, but DNA.  for  Acetabulavia  component.  DNA'  meditewanea  A v e r y minor, f a s t e r  renatur-  t h i s p r o b a b l y r e s u l t s from c o n t a m i n a t i o n  Chlamydomonas  I t i s i n t e r e s t i n g to note t h a t w i t h  c h l o r o p l a s t DNA,  ponents, but  72), but  chloroplast  W e l l s and  Sager (72)  B a s t i a e t a l . (5) r e p o r t o n l y  observe two  k i n e t i c com-  the slower r e n a t u r i n g  component.  Perhaps t h i s d i f f e r e n c e r e f l e c t s d i f f e r e n c e s i n t e c h n i q u e s used f o r ing  the DNA The  and  c a r r y i n g out  the  k i n e t i c c o m p l e x i t y of  renaturation  Acetabulavia  isolat-  reaction.  meditewanea  chloroplast  DNA  9 ( l i l +0.4  x 10  d a l t o n s ) i s comparable to the k i n e t i c c o m p l e x i t i e s  from a number of f r e e - l i v i n g micro-organisms, such as (1.01  + 0.18  x 10  9  daltons),  H. aegyptius  (1.17  + 0.11  Hemophilus x 10  9  o f DNAs  influenzae  daltons),  46  Vasteurella (1.12  muVbooi&a  + 0.02 x 1 0  9  (1.13 + 0.16 x 10  daltons),  Neisseria  9  daltons),  catarrhalisi  Staphylococcus  albus  1.04 + 0.16 x 1 0  9  9 daltons)  (3) and  Acholeplasma  genome s i z e o f 1.1 x 10 1.7 x 10 derable  9  laidlawii  daltons  nucleotide p a i r s .  (1.10 x 10  represents  3.3 x 10  6  T h i s amount o f DNA c o u l d  number o f p r o t e i n s , i n a d d i t i o n to coding  transfer  daltons)  (2). A  nucleotides, or code f o r a c o n s i -  f o r r i b o s o m a l ENA and  RNA.  E x p e r i m e n t a l evidence i n d i c a t e s that c h l o r o p l a s t r i b o s o m a l RNA and p o s s i b l y some t r a n s f e r RNAs a r e s y n t h e s i z e d  mediterranea  and  A. cliftonii  i n the c h l o r o p l a s t s o f A.  (8, 24, 25, 53).  Incorporation  of precur-  s o r s i n t o RNA by i s o l a t e d c h l o r o p l a s t s i s i n h i b i t e d by darkness, a c t i n o mycin and d e o x y r i b o n u c l e a s e ( 8 ) , i n d i c a t i n g t h a t t h i s RNA i s t r a n s c r i b e d from c h l o r o p l a s t DNA. whole c e l l s  and c e l l  Farber's  fragments o f  f i n d i n g s o f Berger ( 8 ) . together  represent  studies  (24,25) o f RNA metabolism i n  Acetabularia  At l e a s t 1.6 x 10  support the  Acetabularia  The r i b o s o m a l RNA f r a c t i o n s found i n  a c h a i n o f about 5 x 10  would be coded by 5 x 10 3  mediterranea  3  3  ribonucleotides  deoxyribonucleotide  (52) .  These  pairs.  DNA base p a i r s a r e r e q u i r e d  t o code f o r t r a n s f e r  RNA, assuming 20 d i f f e r e n t s p e c i e s of c h l o r o p l a s t tRNA each w i t h 80 nucleotides.  This leaves  1,693,400 d e o x y r i b o n u c l e o t i d e  p a i r s (or l e s s i f  the r i b o s o m a l RNAs and t r a n s f e r RNAs are formed from l a r g e r m o l e c u l e s ) f o r r e g u l a t o r y and s t r u c t u r a l genes. is  by  The amount o f DNA which  i n v o l v e d i n the r e g u l a t i o n o f gene a c t i v i t y i s n o t known.  ignored,  precursor  I f this i s  then an approximate maximum number o f p r o t e i n s which can be coded  t h i s amount o f DNA can be e s t i m a t e d .  coded by the c h l o r o p l a s t DNA  (although  I f the ribosomal p r o t e i n s are recent  evidence suggests t h a t  some  47  o f them may  be  under n u c l e a r  a p p r o x i m a t e l y 1.1 This  leaves  16.6  x 10 x 10^  a p p r o x i m a t e l y 5.5 length  of 300  4  amino a c i d s  x 10^  amino a c i d s .  Goffeau and  p a i r s , which c o u l d  c h l o r o p l a s t DNA  c h l o r o p l a s t DNA  Brachet  i f any,  (29)  amino a c i d s i n t o p r o t e i n s by  Aeetabulavia.  are  studied  pairs.  code f o r chain  might code f o r  could  What i s the coded by  experimental evidence c h l o r o p l a s t DNA?  the i n c o r p o r a t i o n of  From the  radioactive  e f f e c t s o f a number o f i n h i b i t o r s on concluded t h a t the s y n t h e s i s  i s dependent on c h l o r o p l a s t DNA.  o f the  the  code f o r most, i f  c h l o r o p l a s t s i s o l a t e d from a n u c l e a t e f r a g -  t h i s p r o c e s s , these r e s e a r c h e r s  t h a t 60%  nucleotide  proteins.  to suggest which p r o t e i n s ,  plast proteins  4  x 10  account f o r  Assuming an average p r o t e i n  a l l , of the c h l o r o p l a s t p r o t e i n s .  ments of  o r 3.3  (70), then the  T h e o r e t i c a l l y , then, the not  (52)  below), they may  deoxyribonucleotide  amino a c i d s  as many as 1800  control—see  Further  of  chloro-  studies  t o t a l r a d i o - a c t i v i t y i s a s s o c i a t e d w i t h the  showed  chloroplast  membranes f o l l o w i n g i n c o r p o r a t i o n o f r a d i o a c t i v e amino a c i d s i n t o by  isolated chloroplasts  (28).  Using a u t o r a d i o g r a p h y , Shephard (56) t i o n and  a c t i n o m y c i n D on  precursors still up  by  compared the e f f e c t s of  the i n c o r p o r a t i o n o f n u c l e i c a c i d and  Aeetabulavia  incorporated  proteins  chloroplasts.  i n t o c h l o r o p l a s t DNA  to 5 weeks a f t e r e n u c l e a t i o n  c h l o r o p l a s t s to i n c o r p o r a t e  had  He  found t h a t  enuclea-  protein  thymidine  was  one week a f t e r e n u c l e a t i o n .  little  e f f e c t on the  uridine i n t o ribonuclease  ability  Periods  of  the  removable m a t e r i a l .  Treatment w i t h a c t i n o m y c i n , however, r e s u l t e d i n the almost complete sence o f l a b e l l e d u r i d i n e from the p l a s t i d RNA anucleate c e l l s .  Incorporation  o f l e u c i n e by  o f normal as w e l l the c h l o r o p l a s t s was  ab-  as affected  48  v e r y l i t t l e by e n u c l e a t i o n , but treatment reduced  with actinomycin  l e u c i n e i n c o r p o r a t i o n i n normal and  anucleate p l a n t s .  concluded  t h a t r e p l i c a t i o n of the c h l o r o p l a s t DNA  nucleus.  He  p l a s t i d RNA  a l s o suggested  i n whole and the dark.  o f the  the evidence i s s t r o n g t h a t s y n t h e s i s o f  (18) found  anucleate  Shephard  i s independent  and p r o t e i n i s under the d i r e c t i o n of p l a s t i d  C r a i g and Gibor  greatly  DNA.  that photosynthetic a c t i v i t y  Acetabutavia  decreased  c e l l s which had been maintained  Upon r e i l l u m i n a t i o n , however, both whole and  regained photosynthetic a c t i v i t y .  anucleate  cells  Treatment w i t h puromycin t o t a l l y i n -  h i b i t e d recovery of photosynthetic a c t i v i t y , i n d i c a t i n g that p r o t e i n t h e s i s i s necessary f o r this recovery. D,  some r e c o v e r y of a c t i v i t y was  r e i l l u m i n a t i o n but f u l l  synthetic  D u r i n g treatment  observed  with  syn-  actinomycin  d u r i n g the e a r l y s t a g e s o f  r e a t t a i n m e n t of p h o t o s y n t h e t i c a c t i v i t y d i d not  o c c u r i n whole or a n u c l e a t e c e l l s . c h l o r o p l a s t DNA  in  T h i s suggests  i s r e q u i r e d d u r i n g the l i g h t  t h a t t r a n s c r i p t i o n of  induced r e c o v e r y o f  photo-  activity.  These c o n c l u s i o n s c o n f l i c t w i t h evidence p r e s e n t e d by Schweiger and o t h e r s which demonstrates n u c l e a r c o n t r o l over some of the proteins.  Schweiger et_ al_.  chloroplast  (54) s t u d i e d the isozyme p a t t e r n s o f m a l i c  dehydrogenase by a c r y l a m i d e g e l d i s c e l e c t r o p h o r e s i s , and were s p e c i e s s p e c i f i c i n four s p e c i e s o f Dasycladaceae F o l l o w i n g e n u c l e a t i o n , the p o s i t i o n s and  found  (Acetabularia).  r e l a t i v e c o n c e n t r a t i o n s o f the  d i f f e r e n t isozyme bands remained c o n s t a n t .  Interspecific nuclear trans-  p l a n t s , however, changed the isozyme p a t t e r n s of the r e c i p i e n t t h a t of the n u c l e u s donor s p e c i e s .  t h a t they  Thus, i t appears  s p e c i e s to  t h a t m a l i c dehydrogenase,  an enzyme a s s o c i a t e d w i t h the c h l o r o p l a s t s , i s c o n t r o l l e d by the n u c l e u s .  49 In a s i m i l a r s e to f experiments,  Reuter and Schweiger (50) demonstrated  that l a c t i c dehydrogenase, also associated with the c h l o r o p l a s t s , i s coded by t h e c e l l nucleus.,  M a l i c dehydrogenase and l a c t i c  are n o t enzymes w h i c h one expects but r a t h e r w i t h mitochondria. preparations dria.  dehydrogenase  t o find associated with chloroplasts,  I t i s quite possible that the chloroplast  used i n these experiments were contaminated with  mitochon-  (53) . Also using e l e c t r o p h o r e s i s , Apel and Schweiger (1) studied t h e pat-  t e r n s o f a m e m b r a n e p r o t e i n f r a c t i o n f r o m A. calyculus chloroplasts.  a n d A.  meditewanea  T h e p r o t e i n p a t t e r n s w e r e c o m p o s e d o f 8 p e a k s common f o r  both species and 3 peaks which were species s p e c i f i c .  S i x weeks a f t e r  interspecific nuclear transplantation or implantation the protein changed t o that o f t h e nucleus  donor species.  chloroplast proteins a r e nucleus n u c l e a r DNA.  Thus, t h e species  pattern specific  dependent and appear t o be coded b y t h e  This does n o t r u l e o u t t h e p o s s i b i l i t y t h a t t h e peaks w h i c h  a r e common t o b o t h s p e c i e s a r e d u e t o p r o t e i n s w h i c h a r e c o d e d b y c h l o r o p l a s t DNA.  Another p o s s i b i l i t y i s that t h e p r o t e i n s which a r e coded b y  the nucleus  c o n s t i t u t e o u t e r membrane p r o t e i n s , w h i l e t h e i n n e r  p l a s t m e m b r a n e c o m p o n e n t s a r e c o d e d b y c h l o r o p l a s t DNA. used would n o t separate membranes.  i n n e r c h l o r o p l a s t membranes and o u t e r  techniques chloroplast  The i n c o r p o r a t i o n o f r a d i o a c t i v e amino a c i d s i n t o t h e membrane  p r o t e i n f r a c t i o n o f Acetabulavia researchers.  The  chloro-  meditewanea  was a l s o s t u d i e d b y  these  The e l e c t r o p h o r e t i c p a t t e r n o f t h e l a b e l l e d c h l o r o p l a s t  teins consisted o f a t l e a s t 3 peaks.  Both cycloheximide  and  pro-  chlorampheni-  c o l a f f e c t e d t h e i n c o r p o r a t i o n o f r a d i o a c t i v i t y i n t o p e a k s 1 a n d 2, w h i l e  50 c y c l o h e x i m i d e i n h i b i t e d the i n c o r p o r a t i o n i n t o peak 3.  Isolated  plasts incorporated  peaks.  amino a c i d s o n l y i n t o the  first  d i c a t e s t h a t some of the p r o t e i n s i n peaks 1 and  two  t e c h n i q u e s to demonstrate s p e c i e s  Schweiger (37) have used the same s p e c i f i c d i f f e r e n c e s i n the  of c h l o r o p l a s t r i b o s o m a l p r o t e i n s  eliftonii  and  p l a n t s and  synthesized  ribosomes.  More r e c e n t l y , K l o p p s t e c h and  patterns  This i n -  2 are p r o b a b l y s y n t h e -  s i z e d on c h l o r o p l a s t ribosomes whereas peak 3 p r o t e i n s are on c y t o p l a s m i c  chloro-  A. evenulata.  from  electrophoretic  A. meditevvanea,  A.  Again, following i n t e r s p e c i f i c nuclear  t r a n s p l a n t s , the p r o t e i n p a t t e r n s  to those of the n u c l e u s donor s p e c i e s .  The  of the h o s t  species  im-  change  r e s u l t s indicate that  those  c h l o r o p l a s t r i b o s o m a l p r o t e i n s which are s p e c i e s - s p e c i f i c are coded the n u c l e a r  genome.  They do not  t e i n bands which are the s p e c i e s may  be  Ceron and during  coded by  same i n the chloroplast  Johnson (16)  the development o f  analyzed  r u l e out  the p o s s i b i l i t y  t h a t the  electrophoretic patterns  pro-  for a l l 3  DNA.  have s t u d i e d the c o n t r o l o f p r o t e i n  Aeetabulavia.  Aeetabulavia  synthesis  U s i n g zone e l e c t r o p h o r e s i s , they  p r o t e i n s from the s o l u b l e , c h l o r o p l a s t i c and. c e l l  t i o n s of a x e n i c a l l y grown  by  evenulata.  Cells  membrane f r a c -  from v a r i o u s  de-  14 v e l o p m e n t a l stages were l a b e l l e d w i t h ment, the e l e c t r o p h o r e t i c p a t t e r n s remained constant r a t e s of s y n t h e s i s  with respect  C leucine.  of proteins  Throughout d e v e l o p -  from the  soluble  to the number of bands, but  of s e v e r a l p r o t e i n s changed.  Proteins  the  fraction relative  from the membrane  f r a c t i o n , however, showed changes i n both the number o f bands i n e l e c t r o p h o r e t i c p a t t e r n and  i n the r e l a t i v e s y n t h e t i c r a t e s of  protein species.  c e l l s were e n u c l e a t e d ,  A f t e r the  t h e r e was  an  the  various initial  51  decrease i n the o v e r a l l r a t e o f s y n t h e s i s fraction. Enucleation  This returned  of proteins  t o normal l e v e l s by 6 weeks p o s t  d i d not a f f e c t the e l e c t r o p h o r e t i c p a t t e r n  tion proteins.  Analysis  from the s o l u b l e  of soluble proteins  enucleation.  o f membrane f r a c -  from p u r i f i e d c h l o r o p l a s t s  a f t e r t h e i r l a b e l l i n g i n normal and a n u c l e a t e c e l l s , showed t h a t both the s t a i n i n g and a u t o r a d i o g r a p h i c even 4 weeks a f t e r e n u c l e a t i o n . ted and l a b e l l e d  in vitro  patterns  were e s s e n t i a l l y unchanged  When i s o l a t e d c h l o r o p l a s t s were i n c u b a -  some o f the components o f the c h l o r o p l a s t  t e i n p a t t e r n were s y n t h e s i z e d .  From these r e s u l t s i t appears t h a t  prosyn-  t h e s i s o f most o f the p r o t e i n components o f the s o l u b l e and membrane f r a c t i o n s i s n o t under immediate c o n t r o l o f the n u c l e u s . t h a t a l l these p r o t e i n s for  Since i t i s u n l i k e l y  a r e coded by c h l o r o p l a s t DNA, the g e n e t i c  messages  a t l e a s t some of them must be i n the form o f l o n g - l i v e d messenger RNA  from the n u c l e u s .  As these r e s e a r c h e r s  i s o l a t e messenger RNA from  Acetabularia  p o i n t o u t , however, attempts t o have been u n s u c c e s s f u l .  Again,  the r e s u l t s suggest t h a t at l e a s t some of the c h l o r o p l a s t p r o t e i n s a r e coded by c h l o r o p l a s t  genes.  D e s p i t e the l a c k o f e v i d e n c e to show t h a t s p e c i f i c p r o t e i n s by  c h l o r o p l a s t DNA, and d e s p i t e  c h l o r o p l a s t s a r e under n u c l e a r  evidence that some p r o t e i n s  a r e coded  associated  with  c o n t r o l , i t seems unreasonable t h a t 1.1.x  9 10  daltons  o f DNA c o u l d  Is i t , perhaps, p o s s i b l e are  e x i s t i n a c h l o r o p l a s t and n o t code f o r a n y t h i n g . t h a t most, i f h o t a l l ,  of the c h l o r o p l a s t  i n f a c t coded by c h l o r o p l a s t DNA, b u t t h a t the e x p r e s s i o n  plast  genes i s under the c o n t r o l o f the nucleus?  suggested t h i s when he s t a t e d :  of chloro-  Or perhaps some p r o t e i n s  may be coded i n the n u c l e u s as w e l l as i n the c h l o r o p l a s t DNA. (28)  proteins  Goffeau  52  "The f a c t t h a t t h e i n c o r p o r a t i o n b y i s o l a t e d c h l o r o p l a s t s i s not dependent upon the time of e n u c l e a t i o n e x c l u d e s t h e p a r t i c i p a t i o n o f a s t a b l e mRNA o f n u c l e a r o r i g i n i n o u r in vitro s y s t e m : i f s u c h w e r e t h e c a s e one w o u l d h a v e e x p e c t e d a d e c a y o f t h e a c t i v i t y a f t e r long periods of enucleation. This c o n c l u s i o n does not n e c e s s a r i l y m e a n , t h a t in vivo, t h e g e n e s i s a n d r e p l i c a t i o n of c h l o r o p l a s t s does not r e q u i r e the p a r t i c i p a t i o n o f c y t o p l a s m i c riibosomes and o f n u c l e u s - c o d e d mRNA...However, i t seems c l e a r , t h a t a f t e r i s o l a t i o n t h e c h l o r o p l a s t s d i s p l a y o n l y t h e i r own i n d e p e n d e n t p r o t e i n s y n t h e s i s c a p a c i t i e s . In other words, a l l the p r o t e i n s w h i c h a r e l a b e l l e d i n o u r in vitro system m u s t b e u n d e r t h e c o n t r o l o f c h l o r o p l a s t DNA a n d n e c e s s a r i l y synthesized i n s i d e the c h l o r o p l a s t s " . I f some p r o t e i n s a r e c o d e d by g e n e s i n t h e n u c l e u s as w e l l as the c h l o r o p l a s t , the n u c l e a r while  g e n e s may  be  transcribed i n whole  the c h l o r o p l a s t genes are r e p r e s s e d .  e v e r , t h e c h l o r o p l a s t g e n e s may e x p l a i n the observations  become a c t i v e .  f r a c t i o n showed an i n i t i a l d e c r e a s e , but On  c l e a t i o n , may  be  the nucleus.  I t i s also possible, of course,  that f o l l o w i n g enufrom the  t e i n s a r e c o d e d b y t t h e n u c l e a r DNA,  while others  Z e t s c h e (76)  soluble  to n o r m a l l e v e l s by  are unaffected  d u r i n g about 4 weeks a f t e r e n u c l e a t i o n .  in  duplication  are coded by the  and  pro-  chloro-  of nucleate  found that i t  and  increases  From the e f f e c t s of v a r i o u s  and p r o t e i n s y n t h e s i s on the i n c r e a s e  t e n t , he c o n c l u d e d t h a t p r o t e i n s y n t h e s i s  enu-  P e r h a p s some c h l o r o p l a s t  examined the c h l o r o p h y l l content  c e l l s o f Aoetabularia.mediterranea  by  transcribed  t h a t t h e r e i s no  genome.  h i b i t o r s o f RNA  could  t r a n s l a t e d f r o m l o n g l i v e d m e s s e n g e r RNA  of c h l o r o p l a s t genes i n the n u c l e a r  anucleate  Such a phenomenon  the o t h e r hand, the membrane f r a c t i o n p r o -  t e i n s w h i c h show changes d u r i n g d e v e l o p m e n t , but  p l a s t DNA.  how-  of proteins returned  cells,  enucleation,  o f C e r o n and J o h n s o n (16)  c l e a t i o n , the o v e r a l l r a t e of s y n t h e s i s  6 weeks p o s t - e n u c l e a t i o n .  Following  in  in-  i n c h l o r o p h y l l con-  i s necessary for chlorophyll  53  formation o r s t a b i l i z a t i o n .  Furthermore, Zetsche concluded t h a t the  c h l o r o p h y l l content o f the c e l l s i s c o n t r o l l e d by c h l o r o p l a s t DNA as w e l l as the n u c l e a r  genome.  I t would be o f i n t e r e s t t o study the DNA:  DNA h y b r i d i z a t i o n between the n u c l e a r  and c h l o r o p l a s t DNA t o determine  the e x t e n t to which they c o n t a i n base sequences i n common, i f a t a l l . As  a l r e a d y mentioned, another a l t e r n a t i v e i s t h a t a l l o f the c h l o r o -  plast proteins  are coded by the c h l o r o p l a s t genome, but the t r a n s c r i p t i o n  of c h l o r o p l a s t DNA i s under n u c l e a r o f c h l o r o p l a s t gene e x p r e s s i o n  control i n intact plants.  might be mediated by s m a l l m o l e c u l e s , the  metabolism o f which i s under d i r e c t n u c l e a r (58)  control.  Shephard and B i d w e l l  Acetabulavia  have found t h a t i s o l a t e d c h l o r o p l a s t s from  meditewanea  c a r r y out normal p h o t o s y n t h e s i s , as w e l l as t h e b i o s y n t h e s i s pigments, l i p i d s and n u c l e i c a c i d s from i n o r g a n i c p r e c u r s o r s , r a t e s as i n t a c t c e l l s .  Control  of proteins, a t the same  They suggest t h a t the membrane s u r r o u n d i n g the  c h l o r o p l a s t s i s h i g h l y s e l e c t i v e and t h a t  in vivo  control of chloroplast  a c t i v i t y may be mediated by t r a n s p o r t mechanisms which govern the r a t e o f e n t r y o f some m o l e c u l e s , such as HCO^, and the r e l e a s e o f products.  T h e i r s t u d i e s a l s o suggest t h a t i n d o l e a c e t i c a c i d may mediate  a c o n t r o l mechanism i n i n t a c t  cells.  O b v i o u s l y , much more r e s e a r c h the  photosynthetic  complex n u c l e a r - c y t o p l a s m i c  Acetabulavia  i s required  i f we a r e t o understand  i n t e r a c t i o n s i n Acetabulavia.  c h l o r o p l a s t s e x h i b i t autonomous b e h a v i o r  Although  in vitvo,  a c t i o n s between the n u c l e u s , the c h l o r o p l a s t s and o t h e r c e l l u l a r must be o f some b e n e f i t t o t h e organism, o t h e r w i s e t h e r e believe  that  Acetabulavia  meditewanea  the i n t e r components  i s no r e a s o n t o  would have e v o l v e d i n t o the complex  54  cell  that i t i s . To  conclude,  Acetabulavia  meditewanea  c h l o r o p l a s t DNA  has  a  9 k i n e t i c c o m p l e x i t y of at l e a s t 1.1 t h i s amount of DNA proteins.  At  could  + 0.4  code f o r many, i f not  the p r e s e n t time, however, there  to i n d i c a t e s p e c i f i c a l l y which p r o t e i n s genome.  x 10  are  daltons.  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