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Isolation and partial characterization of a virus-like particle from a eucaryotic alga Cole, Anabel 1978

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I S O L A T I O N AND  P A R T I A L C H A R A C T E R I Z A T I O N OF  A V I R U S - L I K E P A R T I C L E FROM A E U C A R Y O T I C  ALGA  by ANABEL B.A.,  COLE  Reed C o l l e g e , 1973  A T H E S I S SUBMITTED  I N P A R T I A L F U L F I L L M E N T OF  THE R E Q U I R E M E N T S  FOR THE DEGREE  DOCTOR OF  OF  PHILOSOPHY  in THE F A C U L T Y OF GRADUATE S T U D I E S  We  (Department  of Plant  accept  thesis  to  this  the required  Science)  as  standard  THE U N I V E R S I T Y OF B R I T I S H June,  conforming  COLUMBIA  1978-  | j . A n a b e l C o l e , 197 8  In p r e s e n t i n g t h i s  thesis  an advanced degree at  further  fulfilment  of  the  requirements  the U n i v e r s i t y of B r i t i s h Columbia, I agree  the L i b r a r y s h a l l make i t I  in p a r t i a l  freely  available  for  this  thesis  f o r s c h o l a r l y purposes may be granted by the Head of my Department  of  this  thesis for  It  financial  The  of  Plant  gain s h a l l not be allowed without my  Date  Science  U n i v e r s i t y o f B r i t i s h Columbia  2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  19 May 1978  or  i s understood that copying or p u b l i c a t i o n  written permission.  Department  that  reference and study.  agree t h a t p e r m i s s i o n for e x t e n s i v e copying o f  by h i s r e p r e s e n t a t i v e s .  for  ABSTRACT Large i c o s a h e d r a l v i r u s - l i k e p a r t i c l e s  (VLP's) have  been i s o l a t e d and p a r t i a l l y c h a r a c t e r i z e d from a e u c a r y o t i c f r e s h w a t e r green a l g a .  The a l g a l h o s t , Uronema gicras. was  r e p o r t e d t o c o n t a i n VLP's i n an e a r l i e r u l t r a s t r u c t u r a l  study.  S u b c u l t u r e s o f t h i s a l g a were found t o r e l e a s e VLP's i n t o t h e l i q u i d medium i n w h i c h t h e a l g a was grown, from w h i c h the VLP's were p u r i f i e d w i t h o u t h o m o g e n i z a t i o n o f i n t a c t  cells.  The VLP's i n s i t u w i t h i n i n f e c t e d c e l l s , o r when p u r i f i e d and f i x e d i n g l u t a r a l d e h y d e , have a d i a m e t e r o f approximately  400 nm.  S e c t i o n e d VLP's e x h i b i t an e x t e r n a l  membrane-like s h e l l e n c l o s i n g some r e g i o n s o f d e n s e l y - s t a i n i n g , homogeneous m a t e r i a l and o t h e r r e g i o n s o f f i b r i l l a r m a t e r i a l . About 10% o f t h e p a r t i c l e s a r e t a i l e d , t h e t a i l s measure up t o one m i c r o n i n l e n g t h and a r e a t t a c h e d t o one v e r t e x of the icosahedron.  The development o f the VLP's appears t o i n v o l v e the  n u c l e u s , and a l g a l c e l l s show e x t e n s i v e d i s o r g a n i z a t i o n o f a l l c e l l u l a r membrane systems as the f o r m a t i o n o f t h e VLP's progresses. algal  T a i l e d p a r t i c l e s have been observed f o r m i n g w i t h i n  cells. The VLP's a r e n e v e r r e l e a s e d i n h i g h c o n c e n t r a t i o n s ,  so a l l b i o c h e m i c a l c h a r a c t e r i z a t i o n s have been hampered by t h e l i m i t e d amount o f m a t e r i a l . a d e n s i t y of approximately  P u r i f i e d VLP's from U.. g i g a s have 1.32 g/ml i n s u c r o s e , a s e d i m e n t a t i o n  c o e f f i c i e n t o f 6300 S, and a r e h i g h l y l i g h t s c a t t e r i n g .  The  p o l y p e p t i d e s from t h e VLP's were a n a l y z e d by S D S - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . Ten p r o t e i n bands were d e t e c t e d ; t h e major s p e c i e s had a m o l e c u l a r w e i g h t of 45,000 d a l t o n s .  The n u c l e i c  a c i d c o n t a i n e d w i t h i n the i n t a c t VLP was determined t o be s t r a n d e d DNA  by the f o l l o w i n g methods:  double-  a c r i d i n e orange s t a i n i n g ,  d i p h e n y l a m i n e and o r c i n o l t e s t s , and s p e c i f i c enzyme d i g e s t i o n s of VLP p e l l e t s p r e p a r e d f o r e l e c t r o n m i c r o s c o p y . from the V L P s was 1  Purified  DNA  found t o have a buoyant d e n s i t y o f 1.719  g/ml  i n cesium c h l o r i d e , c o r r e s p o n d i n g t o a molar f r a c t i o n of c y t o s i n e p l u s guanine of about 6 0 % .  The DNA  appeared  t o be  l i n e a r and d o u b l e - s t r a n d e d u s i n g e l e c t r o n m i c r o s c o p i c a l t e c h n i q u e s . Length measurements of the DNA  were v a r i a b l e , r e p r e s e n t i n g 6  DNA  m o l e c u l a r w e i g h t s of 8 x 10  the a c c u r a c y of the t e c h n i q u e was  6  t o 72 x 10  d a l t o n s , although  c o n f i r m e d w i t h DNA's of known  lengths. A l g a l g e r m l i n g s seemed t o be the most s u s c e p t i b l e stage i n the l i f e c y c l e of U. g i g a s f o r s y n t h e s i s and r e l e a s e of VLP's.  No p a r t i c l e s were observed i n t h i n s e c t i o n s of  e l o n g a t e d c e l l s of the mature f i l a m e n t .  Scanning  electron  m i c r o s c o p i c a l views o f l a r g e g e r m l i n g p o p u l a t i o n s showed t h a t a c e r t a i n p e r c e n t a g e of the c e l l s c o n t a i n e d opaque spheres o f a d i a m e t e r s i m i l a r t o the VLP, and by x - r a y m i c r o a n a l y s i s t h e s e spheres were found t o c o n t a i n more phosphorus than the s u r r o u n d ing a l g a l cytoplasm.  A heat shock of 38 C a d m i n i s t e r e d i n the  d a r k , d u r i n g the p e r i o d of zoospore s e t t l i n g , seemed t o g r e a t l y i n c r e a s e the y i e l d o f VLP' s.  Every c u l t u r e o f TJ. g i g a s examined  c o n t a i n e d VLP's; a d e m o n s t r a t i o n of the i n f e c t i v i t y of t h i s  VLP  has not been p o s s i b l e w i t h o u t a h e a l t h y c u l t u r e . - S t r u c t u r a l and b i o c h e m i c a l a s p e c t s of t h e VLP U. g i g a s are u n u s u a l .  from  The p a r t i c l e does not appear t o be  t o any e x i s t i n g group of v i r u s e s ,  related  iv TABLE OF CONTENTS Page ABSTRACT  i i  LIST OF TABLES  ...vii  LIST OF FIGURES  viii  ACKNOWLEDGEMENTS  X  LITERATURE REVIEW  1  MATERIALS AND METHODS  17  Uronema gigas — sources and c u l t u r e techniques  17  C h l o r o p h y l l ,a_ d e t e r m i n a t i o n  18  Virus p u r i f i c a t i o n  20  I. II. III.  Uronema g i g a s VLP  20  Tipula iridescent virus  22  Cauliflower  23  mosaic v i r u s  E l e c t r o n microscopy  24  Scanning e l e c t r o n microscopy  27  Light scattering properties  of UGV  28  Sedimentation c o e f f i c i e n t of UGV  29  Measurements of UGV dimensions  29  Acridine  30  orange s t a i n i n g . . , '  Diphenylamine and o r c i n o l t e s t s  31  Protein electrophoresis  32  Lipids  34  DNA i s o l a t i o n and p u r i f i c a t i o n  36  Buoyant d e n s i t y  37  determinations  E l e c t r o n microscopy of DNA  39  R a d i o i s o t o p e l a b e l l i n g of UGV n u c l e i c a c i d  40  Heat shock experiments  42  V  Page RESULTS I.  II.  44 Interaction A.  Morphology  B.  Cytopathology  C.  Scanning  Isolation A.  III.  o f UGV w i t h  Uronema  gigas  o f UGV a n d i t s h o s t  alga  electron  microscopy  of the host  of germlings  54  P u r i f i c a t i o n o f t h e VLP ' s  C.  Heat  effects  characterization  55  on VLP y i e l d o f UGV  63  and i t s  components  66  A.  Dimensions  B.  Electron of  o f UGV  66  microscopy  pelleted  a n d enzyme  digestion  VLP ' s  scattering  68  C.  Light  D.  Sedimentation  E.  Density  F.  UGV p r o t e i n s  G.  Nucleic  acid  characterization  81  H.  Nucleic  acid  purification  83  I.  Buoyant  density  J.  Nucleic  acid  K.  correction  coefficient  i n sucrose  o f UGV  o f UGV-DNA  75 75  84  v i s u a l i z a t i o n by t h e technique  Radioisotope  labelling  Introduction..  73  78  Kleinschmidt  DISCUSSION I.  50 54  alga  B.  shock  44 48  a n d p u r i f i c a t i o n o f UGV  Growth  Partial  44  87 o f UGV-DNA........  91 96 96  vi Page II. III. IV. V.  Morphology of the p a r t i c l e  96  I n t r a c e l l u l a r development  102  P u r i f i c a t i o n and c h a r a c t e r i z a t i o n  106  Biological  117  LITERATURE CITED  considerations  122  -viiLIST OF TABLES TABLE  PAGE  I  Reported v i r u s - l i k e p a r t i c l e s i n e u c a r y o t i c a l g a e .  II  C h a r a c t e r i s t i c s of single c e l l i s o l a t e s of  5  Uronema gigas  13  III  Composition  19  IV  E l e c t r o n m i c r o s c o p i c counting technique  of B e i j e r i n c k ' s m i n e r a l medium f o r assay  of VLP c o n c e n t r a t i o n V  Release growth  56  of UGV p a r t i c l e s i n r e l a t i o n t o a l g a l (measured as c h l o r o p h y l l a content p e r ml  of c u l t u r e medium) VI  56  Comparison of c o n c e n t r a t i o n and p u r i f i c a t i o n methods of UGV  58  VII  C e l l d i s r u p t i o n methods assayed  f o r JJ. gigas  VIII  Heat shock e f f e c t s on UGV r e l e a s e  64  IX  Measurements of the UGV p a r t i c l e  67  X  Sedimentation  77  XI  UGV p o l y p e p t i d e s d e t e c t e d by SDS-polyacrylamide  c o e f f i c i e n t of UGV  64  gel electrophoresis  77  XII  Nucleic acid colorimetric tests  82  XIII  Buoyant d e n s i t y of UGV-DNA  86  XIV  Guanine p l u s c y t o s i n e molar f r a c t i o n of UGV-DNA..  86  XV  C h a r a c t e r i z a t i o n of UGV n u c l e i c a c i d  XVI  Comparison of the p r o p e r t i e s of ICDV's, cyanophages, and UGV  109  ,  116  -viiiLIST OF FIGURES FIGURE 1  PAGE T h i n s e c t i o n s o f h e a l t h y and VLP-containing  cells  examined by t r a n s m i s s i o n e l e c t r o n microscopy 2  45  P u r i f i e d UGV p a r t i c l e s n e g a t i v e l y s t a i n e d , examined by t r a n s m i s s i o n e l e c t r o n microscopy. S i z e comparisons with TMV and TIV  3  46  • T h i n s e c t i o n s of UGV p a r t i c l e s ; t a i l e d  UGV  p a r t i c l e s w i t h i n c e l l s ; e a r l y stages of i n f e c t i o n with UGV 4  47  Scanning e l e c t r o n microscopy of U_. gigas  germlings;  x-ray m i c r o a n a l y s i s of a l g a l cytoplasm and p o s s i b l e UGV p a r t i c l e s 5  52  Growth of U. gigas and r e l e a s e o f UGV  particles  i n two c u l t u r e media, B e i j e r i n c k ' s and B r i s t o l ' s .  53  6  F i n a l p u r i f i c a t i o n procedure f o r UGV p a r t i c l e s . . .  60  7  Sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n of UGV and TIV  8  '  Enzyme d i g e s t i o n treatments  62 of embedded UGV  p a r t i c l e s : pronase, RNAse, and DNAse 9  Sectioned p e l l e t s o f UGV p a r t i c l e s , p r e - t r e a t e d with o r g a n i c s o l v e n t s  10  L i g h t s c a t t e r i n g c o r r e c t i o n curve  72 f o r one  p r e p a r a t i o n of UGV p a r t i c l e s 11  74  E q u i l i b r i u m c e n t r i f u g a t i o n of UGV, TIV, and BSMV i n sucrose  12  70  Polyacrylamide polypeptides  76 g e l e l e c t r o p h o r e s i s o f UGV 79  -ix-  FIGURE 13  PAGE Microdensitometer scan of g e l shown i n F i g u r e 12  14  Equilibrium  80 centrifugation  DNA's i n cesium c h l o r i d e 15  of UGWDNA w i t h marker  and cesium s u l f a t e  Molecules of DNA from UGV and CaMV v i s u a l i z e d by the K l e i n s c h m i d t technique  16  85  89  D i s t r i b u t i o n of lengths of DNA molecules measured of UGV-DNA and CaMV-DNA, v i s u a l i z e d by the K l e i n schmidt technique  17  Attempted r a d i o i s o t o p e acid  90 l a b e l l i n g of UGV n u c l e i c 94  -xACKNOWLEDGEMENTS  I would l i k e t o e x p r e s s my g r a t i t u d e t o t h e f o l l o w i n g p e o p l e who have g i v e n me a d v i c e and h e l p i n t h e p r e p a r a t i o n o f this thesis.  Dr. J . A. Dodds, who i n i t i a t e d t h i s r e s e a r c h  p r o j e c t and g e n e r o u s l y shared i t w i t h me, has been a c o n t i n u i n g source o f i n t e l l e c t u a l s t i m u l a t i o n .  I am v e r y g r a t e f u l t o Dr.  M a r v i n W e i n t r a u b , D i r e c t o r , A g r i c u l t u r e Canada Research  Station,  Vancouver, f o r t h e use o f t h e e x c e l l e n t f a c i l i t i e s a t h i s s t a t i o n and f o r h i s i n t e r e s t i n my p r o g r e s s . Many members o f t h e s t a f f o f t h e A g r i c u l t u r e Canada R e s e a r c h . S t a t i o n , Vancouver, have h e l p e d me, g i v i n g o f t h e i r time and e x p e r t i s e ;  i n p a r t i c u l a r , Bea S c h r o e d e r , B i l l  Ronald  and Connie N i c h o l s i n s t r u c t e d me i n e l e c t r o n m i c r o s c o p y , a n a l y t i c a l u l t r a c e n t r i f u g a t i o n , and g e l e l e c t r o p h o r e s i s . e s p e c i a l l y i n d e b t e d t o Wes MacDiarmid, t h e Research photographer, f o r h i s help w i t h the f i n a l  I am  Station  photographs.  The members o f my graduate committee have a l l contributed  h e l p f u l i d e a s over t h e c o u r s e o f t h i s t h e s i s .  I  would l i k e t o thank Dr. A . J . Warren, Department o f M i c r o b i o l o g y , U n i v e r s i t y o f B r i t i s h Columbia  (U.B.C.), Dr. B e v e r l y Green,  Department o f Botany, U.B.C., Dr. J.H, Tremaine, Canada Research S t a t i o n , Vancouver  Agriculture  and Dr. V.C. R u n e c k l e s ,  Department o f P l a n t S c i e n c e , U.B.C., f o r t h e i r a d v i c e . I am e s p e c i a l l y g r a t e f u l t o Dr. R . I , H a m i l t o n , my supervisor,  f o r h i s c o n t i n u i n g g u i d a n c e , encouragement, and  s u p p o r t over t h e t h r e e y e a r s I have spent i n h i s l a b .  -xiI have been f i n a n c i a l l y supported f o r the d u r a t i o n of t h i s p r o j e c t by a Postgraduate F e l l o w s h i p  from the N a t i o n a l  Research C o u n c i l of Canada, which i s a l s o g r a t e f u l l y acknowledged.  -1-  LITERATURE REVIEW  V i r a l i n f e c t i o n s or accumulations of v i r u s - l i k e p a r t i c l e s have been r e p o r t e d i n a l l forms of l i f e , p r o c a r y o t i c and e u c a r y o t i c . Many b a c t e r i o p h a g e s and v i r u s e s o f h i g h e r p l a n t s and a n i m a l s have been i s o l a t e d and e x t e n s i v e l y c h a r a c t e r i z e d , t h e r e s e a r c h i n i t i a l l y m o t i v a t e d by the need t o c o n t r o l d i s e a s e s a f f e c t i n g humans and d o m e s t i c a t e d a n i m a l s and plants.  Taxonomic groups i n which few v i r a l i n f e c t i o n s have  been r e p o r t e d , such as the a l g a e , f u n g i , and p r o t o z o a , p r o b a b l y f u n c t i o n as h o s t s t o as many v i r u s e s as t h e more i n t e n s i v e l y s t u d i e d groups, b u t they s i m p l y have n o t been the f o c u s o f as much r e s e a r c h e f f o r t .  T h i s t h e s i s d e s c r i b e s t h e i s o l a t i o n and  p a r t i a l c h a r a c t e r i z a t i o n o f a new v i r u s - l i k e p a r t i c l e  (VLP)  from a e u c a r y o t i c a l g a , Uronema g i g a s . Cyanophages--the v i r u s e s of p r o c a r y o t i c b l u e - g r e e n algae--were  d i s c o v e r e d i n 1963 w i t h the i d e n t i f i c a t i o n o f  LPP-1 v i r u s from a waste s t a b i l i z a t i o n pond i n I n d i a n a (Safferman and M o r r i s 1963).  U n d e r s t a n d a b l y , sewage t r e a t m e n t  ponds p r o v i d e d t h e f i r s t examples of cyanophages s i n c e they a l l o w f a s t and c o n t i n u o u s growth o f the b a c t e r i a l h o s t s t o p o p u l a t i o n d e n s i t i e s which magnify  fluctuations.  In the years  s i n c e the f i r s t i s o l a t i o n a t l e a s t f o u r d i f f e r e n t t y p e s o f cyanophages have been c h a r a c t e r i z e d , and e x t e n s i v e r e s e a r c h has been done c o n c e r n i n g t h e v i r a l i n f e c t i o n c y c l e , e f f e c t s on h o s t metabolism,  and e c o l o g i c a l i n t e r a c t i o n s .  E x c e l l e n t r e v i e w s of  -2cyanophage r e s e a r c h have been p u b l i s h e d r e c e n t l y Padan and S h i l o 1973,  and Safferman  1973)  understanding these v i r u s e s has been r a p i d M o r r i s 1977). in  in  1972,  and progress i n (Safferman  and  Cyanophages are s i m i l a r to other bacteriophages  s t r u c t u r e and b i o c h e m i s t r y , as one would expect c o n s i d e r i n g  the s i m i l a r i t i e s of t h e i r p r o c a r y o t i c h o s t s . LPP  (Brown  One  group,  the  cyanophages, i n c l u d e s p a r t i c l e s which are p o l y h e d r a l , 60 diameter, w i t h a s h o r t n o n - c o n t r a c t i l e t a i l ,  phages T7 and T3.  A second group  i c o s a h e d r a l p a r t i c l e s , about  67 nm  c o l l a r and a t h i n appendage. bacteriophages  (SM-1  resembling  cyanophages) c o n t a i n s  i n diameter w i t h a s h o r t  The N - l group,  (Bradley 1967) , was  nm  s i m i l a r t o type A  o r i g i n a l l y i s o l a t e d from the  f i l a m e n t o u s Nostoc muscorum, and has an i c o s a h e d r a l c a p s i d w i t h a diameter of 55 nm v e r t e x of the head  and a t a i l  of about  110 nm  (Adolph and Haselkorn 1971).  cyanophage d i s c o v e r e d thus f a r i s AS-1,  attached t o one The  largest  from A n a c y s t i s n i d u l a n s  and Synechococcus cedrorum, w i t h an i c o s a h e d r a l head 9 0 nm i n diameter  and a c o n t r a c t i l e t a i l ,  23 nm wide  approximately  (Safferman e t a l . 1972).  245 nm  long by  A l l f o u r groups c o n t a i n  l i n e a r double-stranded d e o x y r i b o n u c l e i c a c i d  (DNA), of molecular  g  weights between 27 and 62 x 10 The SM-1  d a l t o n s (Padan and S h i l o 1973).  group of cyanophages has a complete  dependence on the  c o n t i n u i n g p h o t o s y n t h e s i s of the host c e l l d u r i n g the  infection  cycle.  such  Cyanophages of the N - l and LPP groups have no  requirement  f o r host p h o t o s y n t h e s i s , moreover, both of these  phage types cause e x t e n s i v e d i s r u p t i o n of the host's p h o t o s y n t h e t i c l a m e l l a e , e a r l y i n the i n f e c t i o n c y c l e ,  limiting  -3-  host  photosynthesis  Shilo  1973).  The  (Sherman and  physiology  Haselkorn  o f AS-1  1970,  Padan  i n f e c t i o n s has  and  not  been  characterized. The eucaryotic  existence  algae  P r o t s e n k o , who (Zavarzina infected attacks  exists that cell  1970's.  published  associated  with  Since  at  the  reports  have d e a l t w i t h t o d a t e no  isolation  and  exceptional  Morris  isolation  higher  least  of  the  a tubular  i s discussed  eucaryotic  formidable  obstacle  disruption  of  microscopy  c o n s t i t u t e s the  the  possibility can  infect  have been cells;  VLP's.  (An  t h a t has  been  e a r l y work on  In  presented a l l cases  observed  e n t i r e evidence  a  A l l studies  According  v i r u s e s has  organization  1.  the  cells  successful  polyhedral particle  virus-  appeared u n t i l  changes i n i n f e c t e d  described  to researchers."  cellular  be  and  particle  algae  separately.)  algal  but  i n Table  (1977), i n comparison with of  Tikhonenko  inclusions in algal  ultrastructural  of  a l g a may  thirteen reports  i s presented  purification  the  f u r t h e r d e s c r i p t i o n of  p u b l i c a t i o n has  instance  characterized and  the  the  a phage w h i c h  Mamkaeva 1 9 7 2 ) ,  No  and  pyrenoidosa  of  observed v i r u s - l i k e  1971  Zavarzina  that  symbiont  describing virus-like  summary o f  only;  wall  (Lemke 1 9 7 6 ) .  particles  early  the  by  the  i n v e s t i g a t i o n of  (Mamkaeva 1 9 6 6 ,  Gromov and  infect  of C h l o r e l l a  c u l t u r e s suggested  for lysis  1966,  i n 1958  Further  a procaryotic c e l l  algal  like  lysis  1964).  Chlorella  Zavarzina  the  postulated  observed  1961,  responsible  still  was  of v i r u s e s which  by  to  Safferman  cyanophages, "a the  electron  f o r the  pathogenicity  -4of t h e s e p a r t i c l e s .  D e v i a t i o n s from normal  cell  u l t r a s t r u c t u r e are always observed i n c e l l s which c o n t a i n VLP's, so the p a r t i c l e s appear t o cause a c y t o p a t h o l o g i c a l c o n d i t i o n , but w i t h o u t p r o o f of i n f e c t i v i t y the p a t h o g e n i c e f f e c t s of VLP s y n t h e s i s w i t h i n the a l g a l c e l l s presumptive.  remain  Throughout t h i s t h e s i s the p a r t i c l e s  studied  w i l l be r e f e r r e d t o as VLP's, t o emphasize the l a c k of i n f o r m a t i o n about p a t h o g e n i c i t y . i s r e f e r r e d t o as UGV  When the p a r t i c l e  studied  (Uronema g i g a s v i r u s ) , i t i s s i m p l y t o  r e f e r s p e c i f i c a l l y t o the h o s t a l g a , r a t h e r than t o i m p l y t h a t the t r u e v i r a l n a t u r e o f t h i s VLP has been demonstrated. The h o s t a l g a e l i s t e d i n T a b l e I- are from d i v e r s e taxonomic groups r e p r e s e n t i n g the g r e e n , brown and r e d a l g a e , and s e v e r a l p l a n k t o n i c and u n i c e l l u l a r f l a g e l l a t e s , from b o t h marine and f r e s h w a t e r e n v i r o n m e n t s .  A l l but one of the VLP's  d e s c r i b e d are p o l y h e d r a l ; p a r t i c l e o u t l i n e i n cross s e c t i o n i s hexagonal o r p e n t a g o n a l , s u g g e s t i n g the 5-3-2 icosahedron  (Home 197 4 ) .  symmetry of an  P a r t i c l e d i a m e t e r s range from 25 nm  (Manton and L e a d b e a t e r 197 4) t o 3 00 nm w i t h p o s s i b l e c l u s t e r s a t 50-60 nm  (Mattox e t al_. 1972) ,  (Lee 1971, Chapman and  Lang 1973, Pearson and N o r r i s s 1974, and P i e n a a r 1976), 100 nm  ( P i e n a a r 1976), 130-170 nm  (Toth and W i l c e 1972,  C l i t h e r o e and Evans 1974, Markey 1974, Moestrup and Thomsen 1974, and P i e n a a r 1976), and 200-240 nm  (Pickett-^Heaps 1972,  Swale and B e l c h e r 1973, Hoffman and S t a n k e r 1975).  Size  measurements a r e approximate s i n c e d i f f e r e n t a u t h o r s have used  TABLE I -- Reported v i r u s - l i k e p a r t i c l e s i n e u c a r y o t i c  algae  C e l l type i n f e c t e d and cytopathology caused ___  Classification  Particle Shape Size  red a l g a , freshwater  polyhedral  50-60 nm  A p i c a l c e l l only; Lee nucleus and cytoplasmic membrances degenerate.  green algae, Uronema gigas StigeocIonium sp. freshwater Coleochaete s c u t a t a R a d i o f i l u m sp.  poiy~ hedral  not stated, 50-300 nm from micro -graphs.  D i s o r g a n i z e d nucleus and cytoplasmic degeneration; c e l l types not s p e c i f i e d .  Mattox e t a l 1972  Oedogonium sp,  green a l g a , freshwater  polyhedral  240 nm  Germlings only; cytoplasm and organelles disrupted.  Pickett-Heap 1972  Chorda tomentosa  brown a l g a , marine  polyhedral  17 0 nm  S e t t l i n g spores; general o r g a n e l l a r degeneration.  Toth and W i l 1972  Porphyridium purpureum  red a l g a , marine  polyhedral  40 nm  Tubular n u c l e a r i n c l u s i o n s ; general organellar degeneration.  Chapman 197 2 1973; Chapman and Lang 1973  Aulacomonas SP.  colorless flagellate, green a l g a relative  polyhedral with tail  Loss of n u c l e a r membrane; disorganized organelles.  Swale and Belcher 1973  Algal  host  Sirodotia tenuissima  200-230 150-200  Reference 1971  C e l l type.infected and cytopathology caused  Classification  Particle Shape Size  Ectocarpus fasciculatus  brown a l g a , marine  polyhedral  17 0 nm  Sporangial c e l l s o n l y ; l o s s of n u c l e a r membrane, VLP's and t u b u l a r inclusions i n nucleus.  C l i t h e r o e and Evans 1974.  Chrysochromulina mantoniae  phytoplankton  polyhedral  22 nm  Disorganized  Manton and Leadbeater 1974.  Pylaiella littoralis  brown a l g a , marine  polyhedral  130-170  Young sporangia only; Markey 1974. nuclear d i s o r g a n i s a t i o n and t u b u l a r i n c l u s i o n s ; degenerating o r g a n e l l e s .  Pyramimonas orientalis  flagellate, green a l g a  polyhedral  160-170  Loss of n u c l e a r membrane.  Platymonas sp.  green a l g a , marine  polyhedral  56-58  VLP's i n nucleus cytoplasmic degeneration.  Chara c o r a l l i n a  green a l g a  t u b u l a r 53 2 nm  Apical c e l l s ; Gibbs e_t a l . c y t o p l a s m i c i n c l u s i o n s 1975. of v i r u s .  polyhedral polyhedral polyhedral  13 0 nm  Degenerating nucleus, Pienaar VLP's i n t r a n u c l e a r ; cytomplasmic accumulation.  100 nm  VLP's i n t r a n u c l e a r .  Algal  host  Hymenomonas c a r terae Micromonas p u s i l l a e Cryptomonas sp.  phytoplankton  65 nm  nucleus.  Reference  Moestrup and Thomsen 197 4. only; Pearson and N o r r i s s 1974.  1976.  Algal  host  Cylindrocapsa geminella  Classification  Particle Shape Size  green a l g a , freshwater  polyhedral  200-230  C e l l type i n f e c t e d and cytopathology caused Loss of n u c l e a r membrane; germlings only.  Reference Hoffman and Stanker 1975.  Other VLP's of s i m i l a r morphology i n f u n g i and protozoans Aphelidium  sp.  phycomycete, green a l g a l parasite.  polyhedral  2 00 nm  Inside l y s i n g fungal protoplasts; c y t o p l a s m i c accumulation .  Schnepf e t a l 1970.  Paramoebidium arcaturn  trichomycete, arthropod parasite.  polyhedral  105-110  Limited to nucleus.  Manier e t a l . 1971.  Naegleria gruberi  amoeboflagellate  polyhedral  100 nm  Nuclear o r i g i n ; t r a n s p o r t to cytoplasm.  Schuster and Dunnebacke, 1971.  Entamoeba histolytica  amoeba  polyhedral  70-75  Development i n perinuclear cytoplasm.  Mattern .et a l . 1972, 1974.  i i  -8-  v a r i o u s f i x a t i o n and s t a i n i n g p r o c e d u r e s and i n c l u d e d i f f e r e n t i n t e r n a l s i z e markers.  The o n l y t a i l e d p a r t i c l e was r e p o r t e d  i n t h e f l a g e l l a t e Aulacomonas (Swale and B e l c h e r 1973). D e s c r i p t i o n s o f t h e VLP's, e s p e c i a l l y t h o s e o v e r 50 nm, a r e remarkably c o n s i s t e n t .  E l e c t r o n m i c r o g r a p h s show a  r i g i d o u t e r s h e l l o f r e c o g n i z a b l y p o l y h e d r a l o u t l i n e , 5- o r 6 - s i d e d , s u r r o u n d i n g a dense c o r e o f h e a v i l y s t a i n i n g m a t e r i a l , which may c o m p l e t e l y f i l l  t h e i n t e r i o r o f t h e s h e l l o r occupy  o n l y a p a r t o f t h e i n t e r n a l space.  The s h e l l has a t r i - l a m i n a t e  appearance, v e r y s i m i l a r t o the c l a s s i c a l u n i t membrane i n c e l l s . In a d d i t i o n t o t h e e l e c t r o n - d e n s e c o r e r e g i o n s ,  fibrillar  m a t e r i a l i s o f t e n seen i n " e m p t i e r " r e g i o n s i n t h i n s e c t i o n s o f the VLP's.  The p a r t i c l e s a r e u s u a l l y found i n a p i c a l c e l l s o f  f i l a m e n t s , i n c l u d i n g t e r m i n a l s p o r a n g i a , o r i n zoospores o r young g e r m l i n g s .  No r e p o r t s o f VLP's from mature e l o n g a t e d  c e l l s o f any a l g a have appeared; perhaps r e f l e c t i n g t h e mode o f t r a n s m i s s i o n of the e u c a r y o t i c a l g a l v i r u s e s  (Andrews  1976).  I f t h e VLP's o c c u r as e x t r a c e l l u l a r i n f e c t i o u s a g e n t s , a d s o r p t i o n o r p i n o c y t o s i s o r e n t r a n c e t h r o u g h wounds would be most l i k e l y i n t h e zoospore which does n o t have a c e l l  wall.  S p o r a n g i a l and a p i c a l c e l l i n f e c t i o n s a l s o suggest a t r a n s m i s s i o n p a t t e r n s i m i l a r t o seed t r a n s m i s s i o n i n h i g h e r p l a n t s :  cells  adapted f o r d i s p e r s a l might be most l i k e l y t o become i n f e c t e d , p r o v i d i n g a means o f s p r e a d i n g t h e i n f e c t i o n a l o n g w i t h  algal  growth. I n t r a c e l l u l a r development  o f t h e VLP's appears t o  i n v o l v e t h e n u c l e u s and t h e c y t o p l a s m .  Virus-like  particles  -9u s u a l l y accumulate i n t h e c y t o p l a s m , b u t a n u c l e a r r o l e i n t h e r e p l i c a t i o n c y c l e i s always i m p l i e d : VLP  by a c y t o p l a s m i c s i t e o f  assembly a t t h e p e r i p h e r y o f t h e n u c l e u s ; by t h e f r e q u e n t  o b s e r v a t i o n o f t h e l o s s o f t h e n u c l e a r membrane; and by t h e presence  o f membranous i n c l u s i o n s i n t h e n u c l e i o f s e v e r a l o f  the i n f e c t e d algae  ( P y l a i e l l a , Ectocarpus, Porphyridium).  The  n u c l e a r i n c l u s i o n s appear t o be l o n g c y l i n d e r s , o f a diameter i d e n t i c a l t o t h a t o f t h e VLP's, and o c c a s i o n a l l y w i t h p o l y h e d r a l ends o f a s i z e and appearance s i m i l a r t o t h e VLP's ( C l i t h e r o e and Evans 1974). An e x c e p t i o n a l v i r u s o f a e u c a r y o t i c a l g a i s t h e 532 nm t u b u l a r p a r t i c l e i s o l a t e d from Chara c o r a l l i n a and S k o t n i c k i e_t a l . 1976).  (Gibbs e t a l . 1975  I n f e c t i v i t y has been demonstrated  f o r t h i s v i r u s , and t h e p u r i f i e d p a r t i c l e has been e x t e n s i v e l y characterized.  The p a r t i c l e i s s e r o l o g i c a l l y r e l a t e d t o  tobacco mosaic v i r u s .  T h i s v i r u s i s u n u s u a l among t h e  e u c a r y o t i c a l g a l VLP's i n i t s shape and c l o s e r e l a t i o n s h i p t o the v i r u s e s o f h i g h e r p l a n t s , perhaps r e f l e c t i n g t h e r e l a t i v e c o m p l e x i t y o f t h e h o s t a l g a Chara as compared t o the lower a l g a e . Four o t h e r r e p o r t s o f VLP's a r e i n c l u d e d i n Table 1, one from a phycomycete p a r a s i t e on a green a l g a l  host  (Schnepf _et a l . 1970), one o f a t r i c h o m y c e t e p a r a s i t e o f an arthropod  (Manier est a l . 1971) , and two from amoebae  and Dunnebacke 1971, and M a t t e r n e t a l . 1972).  (Schuster  A l l three  r e p o r t s d e s c r i b e p a r t i c l e s w i t h a morphology and i n t r a c e l l u l a r development s i m i l a r t o t h e a l g a l VLP's.  On t h e b a s i s o f s t r u c t u r e  and c y t o p a t h o l o g i c a l e f f e c t s , these VLP's may r e p r e s e n t a new v i r u s group found  i n the r e l a t i v e l y p r i m i t i v e  eucaryotes.  -10There i s an e x i s t i n g g r o u p i n g of viruses,  the i c o s a h e d r a l  which have s t r u c t u r e s algal viruses.  well-characterized  cytoplasmic deoxyriboviruses  s i m i l a r t o those of the  polyhedral  S t r u c t u r a l a s p e c t s of ICDV's have been  d i s c u s s e d by S t o l t z by McAuslan and  (1971  and  1973), b i o c h e m i c a l development  Armentrout (1974), and  c h a r a c t e r i s t i c s of  group as a whole were reviewed by K e l l y and The  large  (ICDV's)  group i n c l u d e s  viruses  one  mollusc.  iridescent viruses  v i r u s of f i s h , f r o g v i r u s 3, and  r a n g i n g from 130  nm  an i c o s a h e d r a l  f o r the  The  best described  of i n s e c t s ,  lymphocystis  A f r i c a n swine f e v e r v i r u s .  u n i f y i n g c h a r a c t e r i s t i c s of the v i r u s e s suggested by the name:  Robertson (197 3 ) .  which i n f e c t mammals, amphibians,  r e p t i l e s , f i s h , i n s e c t s , and of the group are the  i n t h i s group are  smaller iridescent viruses  as the v i r a l n u c l e i c a c i d .  p l a n t v i r u s e s which might be mosaic v i r u s and d i a m e t e r ) and  included  The  as  shape, of d i a m e t e r t o 300  f o r l y m p h o c y s t i s v i r u s ; v i r u s assembly i n the c y t o p l a s m , d o u b l e - s t r a n d e d DNA  the  The  two  nm  and higher  i n this group--cauliflower  d a h l i a mosaic v i r u s — a r e  much s m a l l e r  seem t o have c l e a r d i f f e r e n c e s  (50  nm  i n chemical  c o m p o s i t i o n , e s p e c i a l l y l i p i d c o n t e n t ( S t o l t z 1973). F r o g v i r u s 3 and  A f r i c a n swine f e v e r v i r u s  enveloped by a t r u e membrane, and  Robertson 1973).  v i r u s and  Two  d e r i v e d from the h o s t c e l l  insect viruses, Tipula  (Kelly  irridescent  Chironomus i r i d e s c e n t v i r u s , have been shown t o  c o n t a i n a p p r o x i m a t e l y 9% by S t o l t z  are  (1973) t o be  l i p i d ( K e l l y and  located  Vance 1973), p o s t u l a t e d  i n s i d e the hexagonal s h e l l ,  s i n g l e b i l a y e r c o v e r i n g the v i r a l c o r e .  P r e v i o u s workers  d i s p u t e d the presence of l i p i d , a t t r i b u t i n g the  small  as  a  had  quantities  -11detected  to contamination  and p u r i f i c a t i o n .  The  by h o s t membranes d u r i n g e x t r a c t i o n  i n t e r n a l l o c a t i o n o f the l i p i d  r e p o r t s t h a t the v i r u s e s are not e t h e r - s e n s i t i v e . controversy  explains  The  i s r e v i e w e d by K e l l y and Vance (197 3 ) .  The  u l t r a s t r u c t u r a l s i m i l a r i t i e s between ICDV's and  the l a r g e p o l y h e d r a l a l g a l v i r u s e s have been noted by authors (Pickett-Heaps and Evans 1974, The  1972,  Markey 1974,  r e p o r t i n 1972  Toth and W i l c e 1972, and Hoffman and  freshwater  filamentous  Clitheroe  S t a n k e r 1976).  by M a t t o x , S t e w a r t and  c i t e d i n T a b l e 1, d e s c r i b e d VLP's p r e s e n t  several  Floyd,  i n f o u r genera o f  green a l g a , a l l of the o r d e r U l o t r i c h a l e s .  The VLP's were o b s e r v e d by t h i s team of p h y c o l o g i s t s i n the c o u r s e of an u l t r a s t r u c t u r a l survey of the o r d e r ; t h e s e workers had no v i r o l o g i c a l e x p e r t i s e t h e m s e l v e s , and proposed t h a t some of the VLP's c o u l d be p u r i f i e d and workers.  S u b s e q u e n t l y Dr. J.A.  s t u d i e d f u r t h e r by  other  Dodds, p o s t - d o c t o r a l f e l l o w ,  Department of Botany, U n i v e r s i t y of B r i t i s h C o l u m b i a , Vancouver, sent f o r a s u b - c u l t u r e o f the c o l l e c t i o n of Uronema qicras r e p o r t e d by Mattox and co-workers t o c o n t a i n VLP's. U l t r a s t r u c t u r a l e x a m i n a t i o n confirmed and  f r e e p a r t i c l e s were d e t e c t e d  o f the c u l t u r e medium.  the presence of the VLP's,  i n negatively stained aliquots  A s i m p l e VLP  i s o l a t i o n was  i n v o l v i n g f i l t r a t i o n t o remove a l g a l c e l l s f o l l o w e d c e n t r i f u g a t i o n to concentrate  the VLP's.  worked o u t , by  R e l a t i v e amounts of  a l g a l growth per f l a s k or t e s t tube were e s t i m a t e d  by an assay  o f c h l o r o p h y l l a. (Hansmaan 1973) . In subsequent work, Dr. Dodds i s o l a t e d 49 by s i n g l e zoospore t r a n s f e r s , and q u a n t i f i e d VLP  subcultures  r e l e a s e i n each  -12of the i s o l a t e s .  The  q u a n t i t y of VLP's r e l e a s e d from each  i s o l a t e v a r i e d w i d e l y , but a l l s u b c u l t u r e s r e l e a s e d VLP's.  In  the s e a r c h f o r a h e a l t h y i s o l a t e of U. g i g a s a second c o l l e c t i o n of the a l g a r e c e i v e d from the Cambridge U n i v e r s i t y Culture C o l l e c t i o n .  T h i s sample proved t o c o n t a i n VLP's as  w e l l ; on f u r t h e r i n v e s t i g a t i o n we  l e a r n e d t h a t the  Indiana  C o l l e c t i o n had been d e r i v e d by s u b c u l t u r e from the Cambridge Collection  (Dr. J . S t e i n , Department of Botany, U n i v e r s i t y of  B r i t i s h Columbia, p e r s o n a l communication).  To date no  c o l l e c t i o n of t h i s a l g a l s p e c i e s has been d e p o s i t e d  other  i n world  c o l l e c t i o n s ; thus i t appears t h a t a l l known U_. g i g a s c u l t u r e s c o n t a i n VLP's.  A h e a l t h y c u l t u r e — w i t h no V L P ' s — i s  a d e m o n s t r a t i o n of  needed f o r  infectivity.  In o t h e r work, Dr. Dodds observed a c o r r e l a t i o n between growth of v e r y l o n g f i l a m e n t s i n some s i n g l e - c e l l - d e r i v e d c u l t u r e s and reduced VLP contained  yield  (Table I I ) , o t h e r  cultures  o n l y r e l a t i v e l y s h o r t f i l a m e n t s and r e l e a s e d VLP's  i n g r e a t e r amounts. fragmentation  A working hypothesis  was  developed t h a t  of the f i l a m e n t r e s u l t e d from VLP  synthesis  subsequent r e l e a s e of VLP's by i n d i v i d u a l c e l l s a l o n g filament.  and  the  H i g h l y i n f e c t e d f i l a m e n t s would be broken i n t o many  s h o r t fragments.  Three c u l t u r e s which were h i g h - y i e l d e r s of  VLP's and t h r e e l o w - y i e l d e r s were s e l e c t e d and assayed f o r c h l o r o p h y l l a, c o n t e n t .  The  high VLP-yielding c u l t u r e s  contained  l e s s than h a l f as much c h l o r o p h y l l a. as the low y i e l d e r s .  In  o t h e r e x p e r i m e n t s Dr. Dodds determined t h a t a c u l t u r e p e r i o d of 6-8  weeks was  1975) .  n e c e s s a r y f o r maximal y i e l d of VLP's (Dodds e t a l .  -13-  TABLE I I .  C h a r a c t e r i s t i c s of s i n g l e c e l l Uronema cricras  i s o l a t e s of  a  C u l t u r e appearance' Fragmented Long f i l a m e n t s 3  VLP index Greater than 5 Less than 5  20/23 3/23  5/26 21/26  Data from J.A. Dodds, p e r s o n a l communication. T o t a l of 49 s i n g l e c e l l i s o l a t e s .  -14-  Uronema g i g a s i s a U l o t r i c h a l e a n green a l g a , of l o n g unbranched f i l a m e n t s , w i t h a s p e c i a l i z e d b a s a l h o l d f a s t c e l l and a p o i n t e d or accuminate t e r m i n a l c e l l .  E x c e p t f o r the  presence of these two d i s t i n g u i s h i n g types of c e l l s , a l l s p e c i e s of Uronema are v i r t u a l l y i d e n t i c a l t o s p e c i e s of U l o t h r i x (Ramanathan  1964).  Taxonomists have d i s a g r e e d about the  validity  of s e p a r a t i n g Uronema from U l o t h r i x on t h i s b a s i s a l o n g 1956),  (Fritsch  but the c h a r a c t e r i s t i c s appear s t a b l e ( a l t h o u g h w i t h some  f l u c t u a t i o n i n response t o d i f f e r e n t e n v i r o n m e n t a l  conditions),  and i n g e n e r a l the two g r o u p i n g s have been approved 1 9 6 4 ) . JJ.  g i g a s was  (Ramanathan  c o l l e c t e d once, i n S w i t z e r l a n d i n  by  1933  V i s c h e r , growing on the s u r f a c e of a s m a l l t r o u g h i n the B o t a n i c a l Gardens i n B a s e l . The  s p e c i e s seems t o grow b e s t i n  s t i l l or o n l y s l i g h t l y moving w a t e r , w i t h o p t i m a l growth i n s p r i n g and f a l l  (Vischer  1933  as r e p o r t e d by Ramanathan  A s e x u a l r e p r o d u c t i o n by f r a g m e n t a t i o n  1964).  i s r e p o r t e d l y l e s s common  than i n o t h e r members of the U l o t r i c h a l e s , but a l l s p e c i e s of Uronema r e a d i l y produce and r e l e a s e a s e x u a l z o o s p o r e s ,  induced  by changes i n the environment r a t h e r than as a c u l m i n a t i o n of c e l l u l a r maturity  (Mitra  1945,  1947).  The b a s a l c e l l and  the  t e r m i n a l c e l l , as w e l l as the c y l i n d r i c a l c e l l s of the f i l a m e n t , can r e l e a s e the q u a d r i f l a g e l l a t e zoospores. t h a t continuous  Mitra  (1947)  suggests  c y c l e s of zoospore p r o d u c t i o n account f o r the  presence of many s h o r t f i l a m e n t s , r a t h e r than d i r e c t fragmentation  of the f i l a m e n t s .  V e r y l o n g f i l a m e n t s of up  to  30 cm can be found i n c u l t u r e d Uronemas, but they do not o c c u r i n nature.  Zoospore p r o d u c t i o n i s r e a d i l y induced  i n c u l t u r e by  t r a n s f e r of f i l a m e n t s t o d i s t i l l e d water or f r e s h medium or  -15merely by d i l u t i o n of the e x i s t i n g medium.  Sexual  reproduction  has not been r e p o r t e d f o r any Uronema s p e c i e s . In c o n t r a s t t o the l i m i t e d d i s t r i b u t i o n of U_. g i g a s , y_. c o n f e r v i c o l u m , which d i f f e r s o n l y i n c e l l u l a r d i m e n s i o n s , 3.5  u t o 8 u broad v e r s u s  8 t o 11 u broad  (Ramanathan 1964), i s  found i n Europe, A f r i c a , the A m e r i c a s , and A s i a , growing e p i p h y t i c a l l y on o t h e r a l g a e and submerged p l a n t s .  Other  members of the genus a l s o have w o r l d - w i d e d i s t r i b u t i o n s . In August 1975  I began r e s e a r c h on the VLP  from y_.  g i g a s w i t h the p l a n of c o n c e n t r a t i n g on i t s b i o c h e m i c a l  and  b i o p h y s i c a l c h a r a c t e r i z a t i o n , r a t h e r than a n a l y z i n g b i o l o g i c a l a s p e c t s of the i n t e r a c t i o n w i t h the h o s t a l g a .  I t was  clear  t h a t l i m i t a t i o n s on what c o u l d be done would be imposed by m i n i m a l amount of VLP's a v a i l a b l e f o r e x p e r i m e n t a t i o n .  the  The  f i r s t a r e a of study i n v o l v e d f i n d i n g an improved p u r i f i c a t i o n technique..  The major d i f f i c u l t y  w i t h the s i m p l e f i l t r a t i o n  c o n c e n t r a t i o n p r o c e d u r e t h a t had been used was a l l p u r i f i e d p r e p a r a t i o n s , of dense s p h e r e s , i n diameter,  was  the p r e s e n c e , i n  about 100-130 nm  which were almost i d e n t i c a l t o the VLP's i n d e n s i t y  and were of unknown o r i g i n and c o m p o s i t i o n . problem  and  The  other obvious  the l i m i t e d amount of VLP's produced even i n the  highest y i e l d i n g cultures.  Some b i o c h e m i c a l methods would be  u s e f u l o n l y when done i n c o n j u n c t i o n w i t h e l e c t r o n m i c r o s c o p y , because of the l i m i t e d s t a r t i n g m a t e r i a l . Two  v i r u s e s were s e l e c t e d f o r use as markers i n  e l e c t r o n m i c r o s c o p y and f o r b i o p h y s i c a l c h a r a c t e r i z a t i o n s of UGV:  T i p u l a i r i d e s c e n t v i r u s ( T I V — a n ICDV) and c a u l i f l o w e r  mosaic v i r u s (CaMV).  P r e l i m i n a r y sucrose d e n s i t y g r a d i e n t  a n a l y s i s i n d i c a t e d t h a t TIV  approximated UGV  i n density,  and  c o u l d t h e r e f o r e be used as a v a l u a b l e marker f o r d e n s i t y g r a d i e n t s , as w e l l as f o r comparisons of s t r u c t u r e s , as  discussed  above. CaMV was  i n c l u d e d as an example of a h i g h e r p l a n t v i r u s  which c o n t a i n s  DNA.  The  g e n e r a l o u t l i n e of my  c h a r a c t e r i z a t i o n of the i n t a c t VLP as s e d i m e n t a t i o n  project  included  i n terms of such p r o p e r t i e s  c o e f f i c i e n t , l i g h t absorbing c h a r a c t e r i s t i c s ,  and d e n s i t y ; a n a l y s i s of VLP  components:  type of n u c l e i c a c i d ,  number and m o l e c u l a r w e i g h t of p r o t e i n s , p o s s i b l e presence of lipid;  and  some i n v e s t i g a t i o n of the u l t r a s t r u c t u r e of  i n f e c t e d c e l l and of VLP's i n s i t u and  in vitro.  C h a r a c t e r i z a t i o n s of t h i s k i n d must be done f o r the VLP's of e u c a r y o t i c a l g a e b e f o r e  the  polyhedral  they can be r e l i a b l y  t o an e x i s t i n g group of v i r u s e s or e s t a b l i s h e d as a new  assigned group.  -17-  MATERIAL5 AND Uronema g i g a s — s o u r c e s and  METHODS  culture  techniques  Uronema gigas c u l t u r e s were obtained from the Type C u l t u r e  C o l l e c t i o n , accession  culture studied  number 174,  by Mattox et. a l . (197 2) .  derived  found t h a t the  source of  the  Another c u l t u r e of JJ.  gigas maintained at Cambridge U n i v e r s i t y was on i n v e s t i g a t i o n we  the  Indiana  also obtained,  Indiana c u l t u r e had  from the Cambridge c o l l e c t i o n , so the  two  but  been  cultures  are  g e n e t i c a l l y i d e n t i c a l u n l e s s changes have o c c u r r e d i n c u l t u r e . Both c u l t u r e s r e l e a s e UGV cytopathologies  show s i m i l a r  (Dr. J . A l l a n Dodds, p e r s o n a l communication).  JJ. gigas was ( S t e i n 1973).  p a r t i c l e s , and  The  grown i n B e i j e r i n c k ' s m i n e r a l medium  procedure f o r making t h i s medium i s shown i n  Table I I I . Stock s o l u t i o n s were s t o r e d  i n d e f i n i t e l y at  d i l u t e d medium was  prepared as needed and  autoclaving  C f o r 20 minutes.  at 121  s o l i d i f i e d w i t h 1% agar was s l a n t s f o r storage of  B e i j e r i n c k ' s medium  cultures. s i n g l e zoospore t r a n s f e r  of f r e s h , s t e r i l e medium i n 25 ml  zoospore formation was  by  used f o r a l g a l growth on t e s t tube  Subcultures were begun by 10 ml  sterilized  4C;  c u l t u r e tubes.  e a s i l y induced by one  medium or d i s t i l l e d water. l e n g t h , were e a s i e r to see  night  to  JJ_. gigas  in fresh  Young germlings,'3 to 4 c e l l s i n and  to manipulate under a d i s s e c t i n g  microscope f o r t r a n s f e r through s e v e r a l drops of medium to r i d a c u l t u r e of contaminants, as d e s c r i b e d  by  Stein  (1973).  Stock  c u l t u r e s maintained on agar s l a n t s were "cleaned" every s i x  -18months by i n d u c i n g z o o s p o r e s o v e r n i g h t i n f r e s h medium, w a s h i n g i n d i v i d u a l g e r m l i n g s on t h e f o l l o w i n g d a y by t h e t e c h n i q u e j u s t mentioned,  and t r a n s f e r r i n g t h e g e r m l i n g s t o f r e s h l y p r e p a r e d  a g a r s l a n t s o r s e e d e r t e s t t u b e s c o n t a i n i n g 10 m l o f medium. growth  liquid  The s e e d e r t u b e s c o u l d be u s e d f o r i n o c u l a t i o n o f l a r g e f l a s k s a f t e r two weeks t o a month o f g r o w t h .  t e c h n i q u e was u s e d t h r o u g h o u t a l l c u l t u r i n g  Sterile  operations.  C u l t u r e s w e r e g r o w n a t 20 t o 23 C a t 4800 t o 5400 l u x and a 16:8 h o u r c y c l e o f l i g h t and d a r k .  Unless otherwise noted  w e r e s t a t i o n a r y and no e x t r a a e r a t i o n was  cultures  supplied.  LL» cricras g r o w t h f o r v i r u s p u r i f i c a t i o n was i n one liter to  f l a s k s c o n t a i n i n g 400 m l o f B e i j e r i n c k ' s medium.  thirty  f l a s k s w e r e i n o c u l a t e d a t one t i m e .  Eight  Maximum v i r u s  c o n c e n t r a t i o n was r e a c h e d a f t e r a b o u t two m o n t h s o f g r o w t h , under normal c o n d i t i o n s . of  Short experiments t o t e s t the e f f e c t s  v a r i o u s t r e a t m e n t s on v i r u s r e l e a s e w e r e more  p e r f o r m e d u s i n g 125 m l f l a s k s c o n t a i n i n g c o u l d be c o n t i n u o u s l y s h a k e n .  practically  40 m l o f medium, w h i c h  Growth i s f a s t e r under these  c o n d i t i o n s ; e x p e r i m e n t s c o u l d be t e r m i n a t e d a f t e r two w e e k s . Chlorophyll a determination C h l o r o p h y l l a c o n t e n t v/as u s e d as a m e a s u r e o f t o t a l a l g a l growth  (Hansmaan 1 9 7 3 ) .  A l g a l c e l l s w e r e washed o f f f i l t e r  p a p e r s u s i n g a known v o l u m e o f d i s t i l l e d w a t e r .  Acetone  added t o b r i n g t h e f i n a l  t o 90% a c e t o n e .  If  Millipore  concentration of l i q u i d  f i l t r a t i o n s had been done, t h e f i l t e r  was  plus  a t t a c h e d a l g a l c e l l s were immersed d i r e c t l y  i n t o 90% a c e t o n e .  The a c e t o n e and c e l l s w e r e t h o r o u g h l y m i x e d  and p l a c e d i n a d a r k  -19TABLE ,111. B e i j e r i n c k ' s m i n e r a l medium ( S t e i n 1973) For Stock I —  Add t o 500 ml o f d i s t i l l e d water:  NH N0 4  K HP0 2  0.10 g  4  0.10 g  CaCl .2H 0  0.05 g  4  2  2  2  Add t o 500 ml o f d i s t i l l e d water:  —  KH P0 2  2  6 0 ml  4.53 g  4  Stock I I I — K HP0  100 ml  0.75 g  3  MgS0 '7H 0  II  1000 ml o f medium  Add t o 500 ml o f d i s t i l l e d water:  40 ml  5.30 g  4  Stock IV -- Trace e l e m e n t s , see below  1 ml Add d i s t i l l e d water t o 1000 m l , and a d j u s t pH t o 6.5.  Trace elements -- Add t o 70 ml warmed d i s t i l l e d w a t e r : 1. 00 g  H3B03 CuS0 .5H 0  0. 15 g  Na EDTA  5. 00 g  4  2  a  2  ZnS0 .7H 0  2. 20 g  M n C l . 4H" 0  0. 50 g  FeS0 .7H 0  0. 50 g  CoCl .6H 0  0. 15 g  (NH.)  0. 10 g  4  2  2  4  2  4 6  2  2  2  Mo_0„,.4H„0 7 ^4 2.  Add s a l t s i n t h e o r d e r g i v e n . A l l o w each addition to dissolve c o m p l e t e l y , and a d j u s t t h e pH t o 5.0'with KOH p e l l e t s b e f o r e adding t h e next s a l t . F i n a l volume s h o u l d be 100 m l . A f t e r 2 t o 3 days a t 4 C, t h e pH s h o u l d have r i s e n t o 6.5 and t h e s o l u t i o n i s ready f o r u s e . D i s c a r d i f i r o n has precipitated.  a) 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  -20r e f r i g e r a t o r f o r a p p r o x i m a t e l y 18 h o u r s . Acetone b l a n k s were i n c l u d e d when M i l l i p o r e f i l t e r s were used.  After  the  e x t r a c t i o n the samples were warmed t o room temperature  and  c e n t r i f u g e d a t 3,000 g f o r 10 minutes t o remove d e b r i s . Absorbance a t 660 nm was Lomb S p e c t r o n i c  r e a d f o r each sample i n a Bausch &  20 colorimeter.  C h l o r o p h y l l a_ c o n c e n t r a t i o n s  of the o r i g i n a l volume of c u l t u r e medium were c a l c u l a t e d by equation  the  below:  ... , . . . 0.95 (A,,.. ) (11.6) ( t o t a l volume of s o l v e n t used) ( c h l o r o p h y l l a.) = 6 60 nm o r i g i n a l volume of medium i n l i t e r s where 11.6 equation  i s the e x t i n c t i o n c o e f f i c i e n t of c h l o r o p h y l l a_.  approximates the t r u e absorbance due  This  to c h l o r o p h y l l a  when measured i n p l a n t e x t r a c t s which a l s o c o n t a i n c h l o r o p h y l l s b and  c. (Hansmaan 1973) .  Virus  purification I . Uronema g i g a s VLP  (UGV)  I n i t i a l attempts t o p u r i f y the VLP hampered by t h r e e f a c t o r s :  from U. g i g a s were  the e x t r e m e l y low c o n c e n t r a t i o n  VLP's i n l a r g e volumes of c u l t u r e medium? the presence of dense s p h e r e s , about one  t h i r d the d i a m e t e r of the UGV  i n the c u l t u r e medium, which co-sedimented w i t h UGV c e n t r i f u g a t i o n s t e p s ; and  the tendency of the UGV  aggregate i r r e v e r s i b l y a t pH 5.5 scheme d e t a i l e d below was The  or below.  The  small  particle,  in a l l  p a r t i c l e s to purification  designed to circumvent these f a c t o r s .  dense b o d i e s were p u r i f i e d by two  c y c l e s of  sucrose d e n s i t y gradient c e n t r i f u g a t i o n (conditions below) and  of  described  then i d e n t i f i e d by e l e c t r o n m i c r o p r o b e a n a l y s i s i n  -21the Geology  Department o f U.B.C.  The dense b o d i e s a r e a m i n e r a l  p r e c i p i t a t e c o n t a i n i n g m a i n l y i r o n w i t h magnesium and c a l c i u m ; t h e y seem t o accumulate  as t h e pH o f t h e medium f a l l s below 5.5.  A l g a l c u l t u r e s were f i r s t f i l t e r e d through 35 u b o l t i n g s i l k t o remove l o n g f i l a m e n t s .  The f i l t r a t e was then  passed t h r o u g h Whatman #1 f i l t e r paper which removed most g e r m l i n g s and zoospores.  I n some cases 1.20 y m M i l l i p o r e  membrane f i l t e r s were used f o r t h i s s t e p , w i t h i d e n t i c a l results.  The a l g a l c e l l s were c o l l e c t e d from t h e f i l t e r s and  s i l k and p o o l e d f o r c h l o r o p h y l l a_ d e t e r m i n a t i o n s .  The c l e a r  s t r a w - c o l o r e d f i l t r a t e was 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 10,000 g f o r one hour i n a S o r v a l l GSA r o t o r .  The r e s u l t i n g  was d i s c a r d e d ; t h e p e l l e t was. resuspended  supernatant  i n a s m a l l volume o f a  b u f f e r c o n t a i n i n g 0.05 M sodium c i t r a t e , 0.01 M d i s o d i u m ethylenediamine t e t r a a c e t a t e  (Na EDTA), and 0.1% T r i t o n X-100, 2  t i t r a t e d t o pH 6.0 w i t h 1 M c i t r i c a c i d .  The V L P s u s p e n s i o n  was d i a l y z e d a g a i n s t 2 l i t e r s o f t h i s b u f f e r o v e r n i g h t .  Citrate  and Na EDTA w i l l c h e l a t e i r o n a t p H s between 5.0 and 7.0; t h i s 1  2  s t e p removed t h e dense b o d i e s .  Sometimes complete removal o f  dense b o d i e s r e q u i r e d s e v e r a l days o f d i a l y s i s w i t h b u f f e r changes. Removal o f dense b o d i e s can be judged f a i r l y a c c u r a t e l y judged by eye, as t h e i n t a c t dense b o d i e s g i v e a y e l l o w t i n g e t o t h e V L P suspension. The d i a l y z e d V L P s u s p e n s i o n was then r e - c e n t r i f u g e d a t 10,000 g f o r one hour i n t h e GSA r o t o r ; t h e s u p e r n a t a n t was d i s c a r d e d , and t h e p e l l e t resuspended (hydroxymethyl)-1,3-propanediol to p r e v e n t p a r t i c l e a g g r e g a t i o n .  i n 0.05 M 2-amino-2  ( T r i s ) - H C l b u f f e r a t pH 8.0  -22F u r t h e r p u r i f i c a t i o n was through 10-40% sucrose g r a d i e n t s  accomplished by  i n 0.05  Small volumes of concentrated VLP's on  12 or 36 ml g r a d i e n t s  i n SW  centrifugation  M T r i s - H C l pH  (0.2  to 4.0  41 or SW  27 and  ml)  8.0.  were  layered  centrifuged  at  10,000 g i n a Beckman L5-75 u l t r a c e n t r i f u g e f o r 30 minutes. resolved  gradients  were scanned at 254  nm  i n an ISCO Model  The UA-5  absorbance monitor; absorbance peaks were c o l l e c t e d by hand. Sucrose from the g r a d i e n t  was  p u r i f i e d VLP's were s t o r e d with sodium azide  removed by d i a l y s i s and  at 4C  i n 0.05  or c h l o r b u t a n o l  the  M T r i s - H C l pH  8.0  added as an a n t i b a c t e r i a l  preservative. II.  P u r i f i c a t i o n of T i p u l a i r i d e s c e n t v i r u s  T i p u l a i r i d e s c e n t v i r u s was method of Kalmakoff and  Tremaine  (TIV)  p u r i f i e d following  (1968).  Infected  paludosa l a r v a e were k i n d l y donated by Mr.  the  Tipula  Fred W i l k i n s o n of  the Canada A g r i c u l t u r e Research S t a t i o n , Vancouver; they were o r i g i n a l l y obtained from Dr. B i o l o g i c a l Control, i n t o small pieces was  magnetically  Delemont, S w i t z e r l a n d .  w i t h a s c i s s o r s and  i n a S o r v a l l SS-34 r o t o r .  The  pH  centrifuged The  at 1,000  p e l l e t was  then c e n t r i f u g e d  rpm  The  f o r 10 minutes  discarded.  The  i n a type 30 r o t o r using  p e l l e t s were suspended i n a 0.01 7.4.  l a r v a e were cut  the r e s u l t i n g homogenate  Beckman L5-75 u l t r a c e n t r i f u g e at 20,000 rpm The  I n s t i t u t e of  s t i r r e d i n d i s t i l l e d water at 4C o v e r n i g h t .  r e s u l t i n g suspension was  supernatant was  Claus K a r l of the  the  f o r 30 minutes.  M potassium phosphate b u f f e r ,  F u r t h e r p u r i f i c a t i o n of TIV was  c e n t r i f u g a t i o n through l i n e a r g r a d i e n t s  achieved  by  of 5 to 40%  sucrose i n  phosphate b u f f e r . gradients  Virus  and c e n t r i f u g e d  20 m i n u t e s .  f o r UGV.  Virus  a t 15,000 rpm i n t h e SW  sodium  pH 7.4, and s t o r e d  against  C h i n e s e cabbage  infected with cauliflower  at 4 C with  mosaic v i r u s  (Brassica  mosaic v i r u s  Vancouver.  This  CaMv s t r a i n was  Rothamsted E x p e r i m e n t a l  pestle  (1975).  Station,  (CaMV) were  2h  --and  mottling  were t h e n c e f o r t h  inoculations.  Infected  months w i t h o u t  losing  The and  and was d o n a t e d by D r . A.A.  Harvey  and v e i n  Chinese  developed  cabbage  noticeable  c l e a r i n g on young  u s e d as t h e v i r u s  source  l e a v e s were s t o r e d  for further  frozen  up t o  v i r u s was p u r i f i e d by t h e method o f H u l l ,  (1976),  leaves  four  infectivity.  Eight  compestris) plants  harvested  approximately  and h o m o g e n i z e d  Shepherd,  C h i n e s e cabbage o r T e n d e r g r e e n  (Brassica  chilled  pH 7.2, and C e l i t e  rubbed onto f o u r  t o 3% weeks, t h e p l a n t s  symptoms—chlorotic  isolated at  The l e a v e s i f e r e h o m o g e n i z e d w i t h a m o r t a r and  a d d e d , and t h e homogenate was After  obtained  Research  originally  i n 0.01 M p o t a s s i u m p h o s p h a t e b u f f e r ,  plants.  (CaMV)  pekinensis)  f r o m D r . P.. S t a c e - S m i t h , o f t h e Canada A g r i c u l t u r e  Brunt e t a l .  described,  0.01 M  i n this buffer  P u r i f i c a t i o n of cauliflower  Freeze-dried  the  UA-5  azide.  Ill,  Station,  onto  41 r o t o r f o r  and f r a c t i o n s were c o l l e c t e d as  f r a c t i o n s were d i a l y z e d  phosphate b u f f e r  leaves  (0.2 ml) were l a y e r e d  G r a d i e n t s were s c a n n e d i n t h e ISCO M o d e l  absorbance monitor  added  suspensions  were i n o c u l a t e d  2 5 days l a t e r .  w i t h CaMV and  Infected  i n a Waring b l e n d o r w i t h  potassium phosphate b u f f e r ,  0.75%  mustard  sodium s u l f i t e ,  leaves  were  0.5 M pH 7.2,  using  1 ml b u f f e r p e r gram of l e a v e s .  The s l u r r y was f i l t e r e d  c h e e s e c l o t h and the volume noted.  through  T r i t o n X-100 and urea were  added t o 2.5% and 1 M, r e s p e c t i v e l y ; the mixture was then s t i r r e d o v e r n i g h t a t 4 C.  A low speed c e n t r i f u g a t i o n f o l l o w e d  (6,000 g, 10 min), and the r e s u l t i n g p e l l e t was d i s c a r d e d .  The  supernatant f l u i d was resuspended i n one tenth the o r i g i n a l volume of 0.0 M potassium phosphate b u f f e r pH 7.2 made t o 2.5% T r i t o n X-100 and 1 M urea, and again s t i r r e d o v e r n i g h t a t 4 C. T h i s suspension was c e n t r i f u g e d a t 27,000 rpm f o r 1.5 the  Spinco Type 30 r o t o r .  hours i n  The r e s u l t i n g supernatant f l u i d was  d i s c a r d e d and the p e l l e t s were slowly d i s p e r s e d i n d i s t i l l e d water o v e r n i g h t .  A final  low speed c e n t r i f u g a t i o n was performed  (10,000 g f o r 10 minutes), and the supernatant was c a r e f u l l y c o l l e c t e d f o r sucrose d e n s i t y g r a d i e n t c e n t r i f u g a t i o n .  The  g r a d i e n t s used were 10-40% sucrose i n 0.01 M potassium phosphate pH 7.2 i n Beckman SW 41 tubes.  C o n d i t i o n s of  c e n t r i f u g a t i o n were two hours a t 23,000 rpm i n the Beckman L5-75 u l t r a c e n t r i f u g e . Resolved g r a d i e n t s were scanned a t 254 nm u s i n g an ISCO Model Ua-5 absorbance monitor.  Fractions  containing  v i r u s were c o l l e c t e d manually, d i l u t e d 1:1 w i t h d i s t i l l e d  water,  and p e l l e t e d by c e n t r i f u g a t i o n a t 45,000 rpm f o r one hour i n the Type 65 r o t o r . storage.  The p e l l e t was resuspended i n d i s t i l l e d water f o r  V i r u s y i e l d , determined u s i n g an e x t i n c t i o n  uncorrected f o r l i g h t scatter ^  0 1%  (E *_° = 7, Shepherd 26 0 nm ' -  averaged about 1 mg/100 g f r e s h weight  coefficient 1970), '  leaves.  E l e c t r o n microscopy V i r u s e s were prepared f o r e l e c t r o n microscopy by negative s t a i n i n g or by f i x a t i o n , embedding, and s e c t i o n i n g .  Two n e g a t i v e s t a i n s were used:  2% p h o s p h o t u n g s t i c  a c i d (PTA)  i n w a t e r , pH 7.2, and 2% aqueous u r a n y l a c e t a t e , pH 5,0. 2% PTA was the s t a i n o f c h o i c e , s i n c e u r a n y l a c e t a t e the UGV p a r t i c l e and caused a g g r e g a t i o n to  t h e low pH.  suspension  p l a c e d on a 200 mesh, c a r b o n / c o l l o d i o n - c o a t e d  was then  The f i n a l drop o f s t a i n was  h e l d on t h e g r i d f o r 15 seconds b e f o r e b l o t t i n g w i t h paper.  Conditions of grid-making  was  copper g r i d f o r  Excess s u s p e n s i o n  washed o f f u s i n g 20 drops o f s t a i n .  overstained  o f v i r u s p a r t i c l e s due  One drop o f a V L P - c o n t a i n i n g  10 minutes a t room t e m p e r a t u r e .  Usually  filter  and s t a i n i n g were s t a n d a r d i z e d -  as much as p o s s i b l e t o a l l o w use o f p a r t i c l e - c o u n t i n g as a measure o f v i r u s c o n c e n t r a t i o n .  Negatively stained p a r t i c l e s  were viewed w i t h a P h i l i p s EM300 e l e c t r o n m i c r o s c o p e a t a m a g n i f i c a t i o n o f 14,000 t i m e s ; counts o f t e n l i n e a r t r a n s e c t s of g r i d squares were averaged f o r an a r t i f i c i a l  index o f v i r u s  c o n c e n t r a t i o n , l a t e r c o r r e c t e d t o a 100X c o n c e n t r a t i o n o f o r i g i n a l c u l t u r e medium. detergent  Counts were found t o be i n a c c u r a t e i f  ( T r i t o n X-100 o r SDS) was p r e s e n t d u r i n g i n c u b a t i o n  on t h e g r i d .  Very few VLP's adhered t o t h e g r i d i n t h e  presence of detergent;  e x t e n s i v e d i a l y s i s t o remove t h e d e t e r g e n t  r e s u l t e d i n much h i g h e r e s t i m a t i o n s o f VLP i n d e x . One p r o c e d u r e was used f o r f i x a t i o n and embedding o f a l g a l c e l l s and p u r i f i e d v i r u s e s .  Clumps o f a l g a l t i s s u e o r  p e l l e t e d v i r u s were f i x e d f o r one hour w i t h 5% in  glutaraldehyde  0.1 M p o t a s s i u m phosphate b u f f e r , pH 7.2, r i n s e d twice i n 15  minute changes o f t h e b u f f e r , and p o s t - f i x e d i n 1% osmium t e t r o x i d e i n Palade's  b u f f e r (Palade 1952) f o r a t l e a s t one hour.  Sometimes t h e a l g a l t i s s u e was mixed i n 2% Noble agar a f t e r  osmium f i x a t i o n t o a l l o w e a s i e r h a n d l i n g o f t h e s m a l l ( B i s a l p u t r a e_t a l . 1973) .  cells.  The f i x e d m a t e r i a l was d e h y d r a t e d  t h r o u g h t e n minute s t e p s o f 30%, 50%, 70%, and 95 % e t h a n o l , and  f i n a l l y t h r e e times i n 100% e t h a n o l , f o l l o w e d by two-  f i f t e e n minute changes o f p r o p y l e n e  o x i d e ; t h e n i t was  i n f i l t r a t e d o v e r n i g h t w i t h a 1:1 m i x t u r e  of p r o p y l e n e  oxide  and Epon (1 p a r t Epon A t o 1 p a r t Expon B p l u s 1.5% DMP-30 accelerator)  ( L u f t 1961).  The n e x t day t h e samples were  embedded i n pure Epon and hardened a t 60 C f o r 36 h o u r s . S e c t i o n s were c u t u s i n g g l a s s k n i v e s on a R e i c h e r t OMU-2 ultramicrotome,  t r a n s f e r r e d t o 100 mesh copper g r i d s , and d r i e d .  They were f i r s t s t a i n e d w i t h 5% u r a n y l a c e t a t e i n 50% methanol for  20 m i n u t e s , t h o r o u g h l y  r i n s e d , and then s t a i n e d f o r t e n  minutes w i t h Reynold's l e a d c i t r a t e N NaOH.  (1961) d i l u t e d 1:4 w i t h  0.01  S t a i n e d s e c t i o n s were examined u s i n g t h e P h i l i p s EM200  or EM3 00 e l e c t r o n m i c r o s c o p e s o p e r a t e d were t a k e n on 3 5 mm  a t 6 0 KV.  Photographs  film.  An a l t e r n a t e f i x a t i o n and embedding method was used for  enzyme d i g e s t i o n s t u d i e s .  mixture  Samples were f i x e d i n a 1:1  o f 5% a c r o l e i n and 5% f o r m a l i n , i n 0.1 M phosphate  b u f f e r , pH 7.2, f o r 90 m i n u t e s , e x c e p t f o r those t o be DNAase-digested, which were f i x e d i n 5% f o r m a l i n alone e t a l . 1969).  In l a t e r experiments,  the usual  glutaraldehyde/  osmium t e t r o x i d e double f i x a t i o n was used w i t h s i m i l a r (Kazama and S c h o r n s t e i n 1972). g l y c o l methacrylate Bernard  (1967).  a UV l i g h t source  (Weintraub  results  F i x e d t i s s u e was embedded i n  (GMA), f o l l o w i n g t h e p r o c e d u r e o f Leduc and  The embedding p r o c e d u r e was c a r r i e d out a t 4 C; (315 nm) was used f o r p o l y m e r i z a t i o n of t h e GMA  -27i n the c o l d room over 3 t o 7 days. f o r d i g e s t i o n of s e c t i o n s 1.  The enzyme s o l u t i o n s used  on g r i d s were:  Ribonuclease A--bovine pancreas Type 12A--(Sigma) 0.5 mg/ml i n d i s t i l l e d water, a d j u s t e d t o pH 6.8 w i t h 0.01  2.  N NaOH;  Deoxyribonuclease I — b o v i n e p a n c r e a s — ( S i g m a ) — 0 . 5 mg/ml i n 0.05 M T r i s - H C l , 0.005 M M g C l , pH 6.8; 2  3.  T r y p s i n - - b o v i n e pancreas Type 1--(Sigma) a t 0,6 mg/ml i n 0.05  4.  P r o n a s e — T y p e 6—(Sigma) a t 0.2 mg/ml i n 0.05 M T r i s - H C l pH  5.  M T r i s - H C l pH 8.0;  7.4; and  L i p a s e 448 ( N u t r i t i o n a l B i o c h e m i c a l Company) a t 0.5 mg/ml i n 0.05 M T r i s - H C l pH 7.8.  G r i d s were f l o a t e d on drops of the enzyme s o l u t i o n s a porcelain  spot p l a t e a t 3 7 C; the i n c u b a t i o n  10 minutes t o o v e r n i g h t . the a p p r o p r i a t e  buffer.  Control  i n w e l l s of  time v a r i e d  from  g r i d s were f l o a t e d on drops of  The spot p l a t e was wrapped i n aluminum  f o i l t o prevent e v a p o r a t i o n .  A f t e r the a p p r o p r i a t e  time, the  s e c t i o n s were r i n s e d , and then s t a i n e d with 2% aqueous a c e t a t e f o r one hour a t 6 0 C, followed  uranyl  by Reynold's lead  c i t r a t e f o r 10 minutes. Scanning e l e c t r o n microscopy Young Tj_. gigas germlings were prepared f o r scanning e l e c t r o n microscopy by growth on a d i a l y s i s membrane. newly r e l e a s e d  Ten ml of  zoospores were added t o a beaker c o n t a i n i n g  50 ml  of f r e s h medium, with a s t e r i l e d i a l y s i s membrance adhering t o the  inner  circumference of the beaker.  Many zoospores  settled  -28i n a t h i n l i n e on t h e membrane a t t h e s u r f a c e o f t h e medium. P i e c e s o f t h e membrane w i t h a d h e r i n g  l i n e s of g e r m l i n g s  c o l l e c t e d a f t e r two days o f growth, f i x e d i n 5%  were  glutaraldehyde  f o r one hour, r i n s e d i n 0.01 M phosphate b u f f e r pH 7.2 t w i c e , and d e h y d r a t e d i n t h e normal e t h a n o l s t e p s . t h e n loaded i n t o s m a l l b a s k e t s d r y i n g method  (Cohen 1975).  The samples were  and d r i e d by the c r i t i c a l  point  S m a l l p i e c e s of d i a l y s i s membrane  w i t h a t t a c h e d zoospores were mounted on aluminum stubs and observed w i t h o u t c o a t i n g i n a H i t a c h i S500 scanning microscope w i t h a Tracor-Northern u n i t attached.  NS 880 x-ray  electron  microanalysis  Pounding w i t h a s m a l l hammer p a r t i a l l y r e l e a s e d  v i r u s - l i k e b o d i e s from t h e d r i e d a l g a l c e l l s . taken u s i n g 35 mm f i l m o r P o l a r o i d 52 f i l m . a n a l y z e d u s i n g t h e x-ray m i c r o a n a l y z e r p e r i o d s a t a v o l t a g e o f 2 0 KV.  Photographs were Samples were  f o r 100 second  counting  The s m a l l e s t o b j e c t i v e a p e r t u r e  was used, w i t h t h e d e t e c t o r 3 cm away from t h e specimen. Phosphorus counts were c o l l e c t e d a t 2.02 KeV. L i g h t s c a t t e r i n g p r o p e r t i e s o f UGV The methods o f Noordam  (197 3) were used t o measure  the l i g h t s c a t t e r i n g c h a r a c t e r i s t i c s o f s e v e r a l UGV  preparations.  L i g h t s c a t t e r i s dependent on p a r t i c l e s i z e and p a r t i c l e aggregation,  so i t can v a r y between p r e p a r a t i o n s o r under  d i f f e r e n t c o n d i t i o n s of storage of v i r u s e s .  The Beckman DK-2A  r e c o r d i n g s p e c t r o p h o t o m e t e r was used t o determine absorbance of VLP suspensions  from 230 nm t o 600 nm.  Curves were drawn  r e p r e s e n t i n g t r u e absorbance o f t h e VLP's, w i t h absorbance due t o l i g h t s c a t t e r s u b t r a c t e d , u s i n g Noordam's g r a p h i n g  techniques  and c a l i b r a t i n g o v e r l a y s .  In l a t e r work d i r e c t measurements  of absorbance a t 2 6 0 nm c o u l d be used f o r an approximation o f t r u e VLP c o n c e n t r a t i o n , u s i n g the c o r r e c t i o n f a c t o r determined from these c u r v e s . Sedimentation c o e f f i c i e n t of UGV The sedimentation c o e f f i c i e n t of i n t a c t UGV p a r t i c l e s was determined u s i n g the Beckman Model E. a n a l y t i c a l ultracentrifuge.  Sedimentation runs were a t 4 0 5 9 o r 6 1 6 6 rpm;  photographs were taken a t 2 or 4 minute i n t e r v a l s u s i n g the s c h l i e r e n and  ultraviolet optics.  runs was maintained a t 2 0 C.  The temperature f o r a l l  Determinations were made on  p r e p a r a t i o n s c o n t a i n i n g the h i g h e s t c o n c e n t r a t i o n s of UGV a v a i l a b l e , u s u a l l y about 3 0 0 p a r t i c l e s p e r t r a n s e c t by e l e c t r o n microscopy.  v i r u s suspensions had t o appear  white by eye t o o b t a i n a r e s u l t w i t h the Model E.  bluish-  Suspensions  of TIV a t 0 . 2 mg/ml were always co-sedimented i n a second quartz c e l l . pH 5 . 5 .  C e n t r i f u g a t i o n s were performed a t pH 8 . 0 and  S c h l i e r e n images were photographed  f i l m , w i t h 1 5 second exposure times. were done by the method o f Markham  on P r o f e s s i o n a l  C a l c u l a t i o n s of S Q / 2  W  (1967).  Measurements o f UGV dimensions Dimensions o f v i r u s p a r t i c l e s were determined by measurements from e l e c t r o n micrographs of n e g a t i v e l y preparations.  stained  Mixtures o f TIV a t 0 . 1 5 mg/ml and UGV a t a VLP  index of 6 0 p a r t i c l e s / t r a n s e c t were s t a i n e d u s i n g the standard procedure i n 2 % phosphotungstic a c i d , pH 7 . 2 . The VLP's were photographed  and then p r i n t e d a t a m a g n i f i c a t i o n of 3 2 , 0 0 0 times.  -30P a r t i c l e dimensions were determined using device UGV  (Dietzgen).  measuring  In a second experiment samples of TIV  were mixed w i t h u n i f o r m l y s i z e d l a t e x beads  diameter, Sigma).  uranyl  acetate,  (790  and  nm  Measurements were made at 20,000 times.  Dimensions of g l u t a r a l d e h y d e - f i x e d  Dr.  a map  pH  5.5,  had  UGV,  and  been p r e v i o u s l y  UGV  stained  determined  with by  J . A l l a n Dodds.  A c r i d i n e orange s t a i n i n g The Spendlove  method of Bradley  (1967) was  v i r u s smears.  followed  f o r a c r i d i n e orange s t a i n i n g of  (TMV), T i p u l a i r i d e s c e n t v i r u s  c a u l i f l o w e r mosaic v i r u s suspensions.  to c l e a n g l a s s  by  Suspensions of bromegrass mosaic v i r u s  tobacco mosaic v i r u s  w i t h UGV  (1965) as d e s c r i b e d  (BMV),  (TIV),  and  (CaMV) were prepared f o r comparison  Drops of v i r u s suspensions were a p p l i e d  s l i d e s w i t h m i c r o p i p e t s and  v i r u s smears were f i x e d by  allowed to dry.  The  immersion i n Carnoy's s o l u t i o n  ( g l a c i a l a c e t i c acid/absolute  ethanol/reagent grade c h l o r o f o r m ,  1:6:3) f o r 5  The  to 10 minutes.  smears were then s t a i n e d  5 minutes with a m o d i f i e d M c l l v a i n e ' s of 0.1 1%  a c r i d i n e orange, pH  i n the 0.15  M c i t r i c a c i d , 4 ml 3.8.  of 0.15 The  buffer containing  M Na HPC> , and 2  4  2  l i q u i d , the  6 ml  ml  s l i d e s were thoroughly  same b u f f e r without a c r i d i n e orange, and  M Na HPO^ f o r 15 minutes.  0.1  of rinsed  transferred  a  photomicroscope with an u l t r a v i o l e t l i g h t source; the use 1 and  to  A f t e r shaking to remove excess  s l i d e s were observed i n a darkened room using  e x c i t e r f i l t e r No.  for  b a r r i e r f i l t e r s Nos.  l i g h t beam r e s u l t e d i n an e m i t t i n g  44 and  53  shortwave u l t r a v i o l e t  Zeiss  of  in  the  light  -31of  approximately 257 nm.  Under these c o n d i t i o n s a s i n g l e -  stranded n u c l e i c a c i d w i l l f l u o r e s c e red and double-stranded n u c l e i c a c i d s w i l l f l u o r e s c e green.  I f a red f l u o r e s c e n c e i s  seen, a t e s t can be performed t o d i s t i n g u i s h RNA exposing the smears to 0.1 M A s i n g l e - s t r a n d e d DNA  citric  by  a c i d f o r 1 t o 5 minutes.  w i l l show f a d i n g of the red f l u o r e s c e n c e ,  p o s s i b l y to green; a s i n g l e - s t r a n d e d RNA (Bradley  from DNA  remains r e d - f l u o r e s c i n g  1965). Even w i t h v e r y s u c c e s s f u l s t a i n i n g , the amount of  light  a v a i l a b l e f o r photography i s extremely l i m i t e d u s i n g  t h i s technique.  The microscope i r i s must be completely open,  and the d i f f u s i o n d i s k f i l t e r  at the back of the microscope  must be removed from the l i g h t beam.  Exposure times of up to  two minutes were common, u s i n g Kodak Ektachrome Diphenylamine and o r c i n o l  ASA  160.  tests  The diphenylamine and o r c i n o l t e s t s , which d i s t i n g u i s h r i b o n u c l e i c a c i d from d e o x y r i b o n u c l e i c  acid  (Pederson 1969), were used to determine the type of n u c l e i c of UGV,  f o l l o w i n g the methods of Shatkin  (1969).  t e s t , samples of whole v i r u s were brought to 5% acid at  For e i t h e r trichloroacetic  (TCA), incubated a t 90 C f o r 20 minutes, and  3,000 g f o r 10 minutes.  e x a c t l y as d e s c r i b e d . of UGV  acid  centrifuged  Otherwise t e s t s were performed  E v e n t u a l l y i t become apparent t h a t  and TIV prepared i n t h i s manner gave m i s l e a d i n g  samples  results,  perhaps due to the presence of sugar r e s i d u e s other than those p r e s e n t i n the n u c l e i c a c i d s .  Diphenylamine and  were found t o be more r e l i a b l e when p u r i f i e d v i r a l  orcinol nucleic  tests  -32a c i d s from t h e v i r u s e s were t e s t e d .  The RNA's o f tobacco  mosaic and bromegrass mosaic v i r u s e s were used as RNA DNA from TIV and CaMV were used as DNA s t a n d a r d s . p u r i f i c a t i o n methods a r e d e s c r i b e d  standards;  Nucleic acid  i n a separate section of  M a t e r i a l s and Methods. Protein  electrophoresis V i r u s e s were d i s s o c i a t e d f o r a n a l y s i s o f p r o t e i n  components by a m o d i f i c a t i o n o f a method o f M a i z e l V i r u s samples (24% u r e a ,  (1971).  (1 mg/ml) were mixed w i t h a d i s s o c i a t i o n b u f f e r  1% SDS, and 1% B-mercaptoethanol i n 0.1 M N a H P 0 , 2  4  f r e s h l y prepared) i n a r a t i o o f 3:1 (w/v) . The m i x t u r e was b o i l e d f o r 90 seconds.  A l t e r n a t i v e l y , a s m a l l volume o f VLP's  was made 1 N i n HC1, a l l o w e d overnight,  t o stand a t room temperature  and c e n t r i f u g e d a t 6,000 rpm f o r 15 m i n u t e s ; t h e  p e l l e t was then d i s s o l v e d i n d i s s o c i a t i o n b u f f e r personal  communication).  electrophoresced  (J.H. Tremaine,  D i s s o c i a t e d p r o t e i n s were  i n g e l s c o n t a i n i n g 1% SDS and 5, 7.5 o r 10%  polyacrylamide. Stock s o l u t i o n s f o r p r o t e i n e l e c t r o p h o r e s i s 1) 10E:  1.0 M N a H P 0 / N a H P 0 2  4  2  4  included:  b u f f e r , pH 7.2, 0.1% SDS, w i t h  0.1 ml B-mercaptoethanol added p e r l i t e r .  This  s o l u t i o n was used f o r t h e e l e c t r o p h o r e s i s b u f f e r (IE) a t a 1/10 d i l u t i o n w i t h d i s t i l l e d w a t e r ; i t was used a t 3/10 (3E) f o r t h e g e l b u f f e r , w i t h 3 ml o f 10% SDS added t o each 97 ml o f d i l u t e d s t o c k b u f f e r and Tremaine  (Agrawal  1972).  2) A c r y l a m i d e (Eastman Kodak), 30%, i n d i s t i l l e d w a t e r ,  filtered  -33a f t e r d i s s o l v i n g , and w i t h 0.75% N , N - m e t h y l e n e - b i s 1  acrylamide  (Eastman Kodak) added.  3) TEMED (N,N,N',N'-tetramethyladiamine, Eastman Kodak), 1% in d i s t i l l e d  water.  4) Ammonium p e r s u l f a t e , 10% i n d i s t i l l e d w a t e r , made f r e s h every tv/o weeks ( F r a e n c k e l - C o n r a t  and R u e c k e r t 1967 ,  Maurer 1971) . Twelve g e l s were c a s t u s i n g a t o t a l volume o f 36 ml of g e l m i x t u r e . The  proportions of the mixture  acrylamide  f o r 10% g e l s were 12 ml  s t o c k , 12 ml 3E b u f f e r , 7 ml d i s t i l l e d w a t e r , 0.36 ml  10% SDS, 3.6 ml TEMED, and 0.18 ml 10% ammonium p e r s u l f a t e , added l a s t w i t h m i x i n g .  P l a s t i c g e l tubes were each q u i c k l y  f i l l e d w i t h 3 ml o f t h e m i x t u r e , and d i s t i l l e d water was c a r e f u l l y l a y e r e d over t h e g e l m i x t u r e surface.  t o ensure a l e v e l g e l  A minimum o f one hour was a l l o w e d f o r p o l y m e r i z a t i o n .  When t h e g e l s had s e t t h e tubes were i n s e r t e d i n t o t h e upper t r o u g h o f t h e e l e c t r o p h o r e s i s chamber, which was then f i l l e d 800 ml o f IE b u f f e r .  with  T h i s assembly was lowered i n t o t h e lower  t r o u g h c o n t a i n i n g 1200 ml o f I E b u f f e r .  P r o t e i n samples  (50 t o 200 u l ) i n d i s s o c i a t i o n b u f f e r were made more dense w i t h the a d d i t i o n o f 10% g l y c e r o l c o n t a i n i n g 1% bromophenol b l u e as a marker, and l a y e r e d on t h e g e l s u r f a c e u s i n g a m i c r o p i p e t under the b u f f e r s u r f a c e . for  4.5 h o u r s .  The g e l s were e l e c t r o p h o r e s e d  a t 10 mA/gel  G e l s were removed from g e l tubes by g e n t l e  b l o w i n g o r by f o r c i n g water through a s y r i n g e n e e d l e between t h e i n t e r i o r o f t h e g e l and t h e i n n e r s u r f a c e o f t h e p l a s t i c and were immediately  immersed i n s t a i n .  tube,  The s t a i n used was 0.1%  Coomassie b r i l l i a n t blue R-50 i n 15% a c e t i c a c i d , 50% e t h a n o l . The  g e l s were s t a i n e d o v e r n i g h t a t 37 C and then d e s t a i n e d i n  7% a c e t i c a c i d , 50% e t h a n o l a t 37 C o v e r n i g h t . r e - s w o l l e n and s t o r e d i n 7% a c e t i c a c i d .  The g e l s were  P r o t e i n molecular  weights were determined by p r e p a r i n g a standard p l o t f o r each e l e c t r o p h o r e s i s run, showing d i s t a n c e migrated versus  i n the g e l s  the logarithms of the molecular weights of p r o t e i n  standards,  as d e s c r i b e d by Loening  (1969).  Protein  used were c a r b o n i c anhydrase, myoglobin, ovalbumin, serum albumin, and a l c o h o l dehydrogenase of  standards bovine  (molecular weights  29,000, 17,500, 43,000, 66,000 and 37,000 d a l t o n s ,  r e s p e c t i v e l y — S i g m a Biochemical  Company).  In one i n s t a n c e a s l a b g e l was used i n s t e a d of c y l i n d r i c a l gels.  A 10% p o l y a c r y l a m i d e  separating g e l i n a slab  h o l d e r was capped with an 8% g e l a f t e r p r o t e i n samples had been added t o i n d i v i d u a l w e l l s . cylindrical gels.  B u f f e r s were the same as f o r  Voltage was s u p p l i e d from a Buchler power  supply, with a slow i n c r e a s e i n v o l t a g e from 10 V t o 40 V over the f i r s t hour of e l e c t r o p h o r e s i s ; 40 t o 50 V or 15 0 mA were maintained had  almost reached  over  approximately  3 t o 4 hours u n t i l the dye f r o n t  the g e l end.  Estimates  determined u s i n g t h i s system corresponded  of molecular w e l l with  weights  values  determined u s i n g c y l i n d r i c a l g e l s ; p r o t e i n band r e s o l u t i o n was better using t h i s  system.  Lipids P r e p a r a t i o n s of VLP's were t r e a t e d w i t h ether or .chloroform/methanol  (2:1) i n order t o i n v e s t i g a t e the presence  or absence of l i p i d s i n these p a r t i c l e s .  In the  experiment small a l i q u o t s of VLP's i n storage  first  b u f f e r were  g e n t l y shaken f o r 20 minutes at room temperature w i t h 3 times t h e i r volume of each s o l v e n t . p e l l e t e d at 10,000 g f o r one and  The  e x t r a c t e d VLP's were  hour, and  then f i x e d , dehydrated,  embedded f o r e l e c t r o n microscopy using the  methods.  S e c t i o n s were cut, s t a i n e d , and  standard  examined f o r a l t e r a t i o n s  of virion s t r u c t u r e a f t e r the s o l v e n t treatments. were s e c t i o n s of VLP's p e l l e t e d and  Control  grids  f i x e d without a s o l v e n t  extraction. In a second experiment, s o l v e n t e x t r a c t i o n of VLP's was  f o l l o w e d by  of Tas  slab gel protein electrophoresis.  e t a l . (1977) and Amako and  Yasunaka  f o l l o w e d with s l i g h t m o d i f i c a t i o n s .  The  methods  (1977) were  A small volume  (0.6 ml)  of  concentrated  VLP's was  ether, using  30 volumes of s o l v e n t f o r every volume of v i r u s .  The  chloroform/methanol sample was  minutes and One  then mixed slowly  f o r two  hours on a r o t a r y shaker.  taken up  i n 0.9  concentrated concentrate  then added and  c e n t r i f u g e d at 3,000 g f o r 15 minutes.  electrophoresis.  The  was  the  pellet  ml of p r o t e i n d i s s o c i a t i o n b u f f e r f o r  The  chloroform/methanol supernatant  was  to near dryness u s i n g a r o t a r y evaporator and  ether-treated  taken up i n 0.9  sample was  The  mixed f o r 2 0 minutes at room temperature, 0.3  ml of d i s s o c i a t i o n b u f f e r  added to the remaining aqueous phase.  samples were analyzed  the  ml of d i s s o c i a t i o n b u f f e r .  the ether removed by p i p e t t e , and was  shaken v i g o r o u s l y f o r 5  t h i r d of the t o t a l sample of methanol was  mixture was was  shaken with e i t h e r chloroform/methanol or  as d e s c r i b e d  The  solvent  treated  i n the s e c t i o n on p r o t e i n  -36e l e c t r o p h o r e s i s ; samples were c o - e l e c t r o p h o r e s e d w i t h d i s s o c i a t e d v i r u s t h a t had not been t r e a t e d w i t h o r g a n i c solvents. DNA i s o l a t i o n and p u r i f i c a t i o n DNA was i s o l a t e d from the VLP's o f JJ. g i g a s by the method o f Shepherd  e t al..  (197 0) .  The c o n c e n t r a t i o n o f v i r u s  i n each sample was determined, by absorbance  a t 26 0 nm,  c o r r e c t e d f o r l i g h t s c a t t e r , and brought t o 0.25% (w/v) sodium dodecyl s u l f a t e  (SDS).  Pronase  (1 mg/ml) was added t o a f i n a l  c o n c e n t r a t i o n o f 5 0 ug/mg of v i r u s .  The mixture was incubated  at 37 C f o r 2 hours, then more SDS was added t o a f i n a l c o n c e n t r a t i o n of 1% SDS.  The i n c u b a t i o n was continued f o r one  hour or u n t i l opalescence had disappeared.  The DNA was f u r t h e r  p u r i f i e d by two e x t r a c t i o n s with s a l i n e - s o d i u m - c i t r a t e s a t u r a t e d phenol  (SSC:  (SSC)-  0.15 M Na c i t r a t e , 0.015 M NaCl, pH 7.0).  An equal volume of SSC-saturated phenol was added t o each v i r u s sample,  and the mixtures were g e n t l y mixed on a r o t a r y  at low speed f o r 15 minutes.  shaker  The r e s u l t i n g emulsion was  c e n t r i f u g e d a t 6,000 rpm f o r 10 minutes; the lower phenol  layer  and some denatured p r o t e i n a t the i n t e r f a c e were removed by pipette.  The upper aqueous l a y e r was r e - e x t r a c t e d with phenol.  A f t e r a second c e n t r i f u g a t i o n , the aqueous l a y e r was g e n t l y w i t h e t h e r t o remove t r a c e s of phenol.  shaken  Care was taken  d u r i n g a l l m a n i p u l a t i o n s of the DNA t o minimize s h e a r i n g . F i n a l l y , the DNA was d i a l y z e d a g a i n s t 2 l i t e r s of SSC, o v e r n i g h t at 4 C.  P u r i f i e d DNA was s t o r e d f r o z e n i n s m a l l a l i q u o t s .  DNA of low c o n c e n t r a t i o n s was handled, a l l glassware was  When  p r e - t r e a t e d w i t h " S i l i c l a d , " a commercial c o a t i n g compound, f o l l o w i n g d i r e c t i o n s on t h e package.  Untreated  glassware  b i n d s DNA: S i l i c l a d c o a t i n g forms a h y d r o p h o b i c s u r f a c e and prevents  binding.  T h i s method o f DNA p u r i f i c a t i o n worked  w e l l f o r p u r i f i c a t i o n o f DNA from '..CaMV and TIV as w e l l as from UGV.  Concentration  o f DNA was d e t e r m i n e d by absorbance 2  at 260 nm, u s i n g an e x t i n c t i o n c o e f f i c i e n t o f 20 cm /mg (Shepherd e t a l . 1968). G e n t l e r methods o f o b t a i n i n g DNA i n v o l v i n g o n l y SDS o r mixtures  o f SDS and m i l d p r o t e i n d e n a t u r a n t s  1967 , K e l l y and Avery 1974, the v i r u s .  ( B e l l e t t and Inman  Garwes ejb a l . 1975) d i d n o t d i s r u p t  A m i l d e r d i g e s t i o n u s i n g t h e f u n g a l enzyme  p r o t e i n a s e K (Hansen 1974,  G r o s s - B e l l a r d e t a l . 1973) was n o t  s e v e r e enough t o r e l e a s e t h i s DNA, even w i t h  overnight  incubations. In one i n s t a n c e DNA was p r e p a r e d f o r e l e c t r o n m i c r o s c o p y by b r i n g i n g a s m a l l a l i q u o t o f VLP's t o 0.1 M NaOH, pH 13.0.  A f t e r 2 t o 6 hours a t room temperature e s s e n t i a l l y a l l  i n t a c t VLP's had d i s a p p e a r e d ;  t h e l i b e r a t e d DNA was used  without  further purification. Buoyant d e n s i t y  determinations  Determinations  o f buoyant d e n s i t y were made by  c e n t r i f u g a t i o n o f UGV-DNA i n cesium c h l o r i d e (CsCl) P u r i f i e d DNA a t a p p r o x i m a t e l y  gradients.  5 t o 10 ug/ml i n Tris-EDTA b u f f e r  (0.1 M T r i s , 0.01 M Na EDTA a t pH 8.5) was added t o 0.951 g o f 2  o p t i c a l l y pure C s C l t o b r i n g t o 1 ml t o t a l volume. was  Marker DNA  added (5 t o 10 u l o f 1 t o 2 mg/ml o f DNA from E s c h e r i c h i a  -38c o l i . Micrococcus of C s C l was  l y s o d e i k t i c u s or T I V ) .  The  resulting density  measured a t 25 C u s i n g an Abbe r e f r a c t o m e t e r w i t h a  sodium lamp.  A d d i t i o n a l C s C l was  added, i f n e c e s s a r y  the d e n s i t y of the s o l u t i o n t o a p p r o x i m a t e l y  1.700  to b r i n g  g/ml.  When  cesium s u l f a t e g r a d i e n t s were used, the i n i t i a l d e n s i t y approximately  1.40  g/ml.  C e n t r i f u g a t i o n s were run on a Beckman  Model E a n a l y t i c a l u l t r a c e n t r i f u g e a t 44,000 rpm, t o 22 h o u r s .  was  20 C, f o r 20  P i c t u r e s were taken u s i n g the u l t r a v i o l e t  light  s o u r c e , a t zero t i m e , a f t e r 4 t o 8 h o u r s , and a t the end  of  each r u n , u s i n g Kodak P r o f e s s i o n a l Pan f i l m #4155.  Exposure  s e r i e s of 10 seconds t o 2 minutes were u s e f u l .  negatives  were scanned u s i n g a J o y c e - L o e b e l t o 2.0  O.D.  u n i t wedge.  d e n s i t y were used:  Two  The  microdensitometer  w i t h the 0  methods of c a l c u l a t i o n of buoyant  the r e f e r e n c e DNA  method (Sueoka  1961,  Mandel e_t a l . 196 8) , and the a b s o l u t e method, r e q u i r i n g no marker DNA  (Chervenka 1973).  c y t o s i n e was  The molar f r a c t i o n of guanine p l u s  c a l c u l a t e d u s i n g the e q u a t i o n of S c h i l d k r a u t e t a l .  (1962) . In o r d e r t o prove t h a t the i s o l a t e d n u c l e i c a c i d DNA,  the banded n u c l e i c a c i d was  re-run i n a CsCl gradient. C s C l was  The  DNA  (Worthington) and d i g e s t was was  s o l u t i o n by d i a l y s i s v e r s u s  s o l u t i o n was 0.01  then the SSC  brought t o 100 ug/ml DNAse  M M g C l , and d i g e s t e d f o r two h o u r s .  r e - d i a l y z e d versus  re-added w i t h marker DNA  again to e q u i l i b r i u m .  h y d r o l y z e d w i t h DNAse and  A f t e r a s u c c e s s f u l Model E, run  removed from the DNA  overnight.  was  2  SSC w i t h added EDTA (0.01 M), and  the s o l u t i o n was  The CsCl  centrifuged  -39-  E l e c t r o n microscopy Purified the  technique  o f DNA DNA was p r e p a r e d  of Kleinschmidt  (1968)  D a v i s .et a l . 1 9 7 1 , Younghusband Kaesberg coated  1977).  with  iso-amyl before  Tabbed  a thin  acetate.  use:  prepared  filled  Buchner  t h e g r i d s were l e f t  must be u s e d w i t h i n  All  glassware  five  s o l u t i o n s were p r e p a r e d stock—10  acetate  paper.  The  to dry f o r at least resolution  covered 2 days.  and a r e v e r y  days,  as t h e f i l m  with  "Siliclad."  becomes  clean,  unstable.  The f o l l o w i n g  f o r the Kleinschmidt  technique:  ' c ' i n 10 m l o f 5 M  pH 8.5;  s t o c k — 0 . 2 5 M ammonium  hypophase  3)  Tris-EDTA—0.1 M Tris,  acetate  pH 8.5;  0.01 M N a E D T A pH 8 . 5 . 2  (1 t o 45 u l ) a t 10 t o 50 ug/ml were combined  5 u l o f hyperphase the t o t a l  d i s h was f i l l e d r o d was r u n o v e r A thoroughly  way was  funnel containing a small  mg c y t o c h r o m e  2)  Samples o f DNA  i n this  The f u n n e l was s e c u r e l y  i n place  u s e d was c o a t e d  hyperphase  bring  24 h o u r s a t 60 C  on Whatman #1 f i l t e r  way g i v e h i g h  but  ammonium  3.5% P a r l o d i o n i n  from t h e f u n n e l , a l l o w i n g t h e P a r l o d i o n  on t h e g r i d s .  G r i d s made i n t h i s  1)  from  mesh) were  One d r o p o f P a r l o d i o n p r e p a r e d  t o a water  to settle  stock  (Pelco—22  The P a r l o d i o n was b a k e d  w a t e r was s l o w l y d r a i n e d  and  some m o d i f i c a t i o n s  and L e e 1 9 7 4 , M c C l e m e n t s and  copper g r i d s  number o f c l e a n g r i d s r e s t i n g  film  with  t h e d i s s o l v e d P a r l o d i o n was s t o r e d d e s s i c a t e d i n  a brown b o t t l e . applied  film  f o r e l e c t r o n m i c r o s c o p y by  stock,  and t h e T r i s - E D T A  volume t o 50 u l . with  b u f f e r was added t o  square p l a s t i c  hypophase t o form a h i g h m e n i s c u s .  the p e t r i  cleaned  A 90 mm  d i s h t o c l e a n t h e hypophase  glass slide  resting  with  petri A glass  surface.  on a s m a l l b l o c k o f  p l a s t i c formed a ramp i n t o the hypophase the hypophase. the hypophase  approximately 45° to  A g l a s s c a p i l l a r y p i p e t t e was used t o t r a n s f e r to the top  of the ramp; the hyperphase was allowed  to run down the ramp to the hypophase  s u r f a c e . G r i d s were  touched to the hyperphase s u r f a c e , h e l d i n s t a i n 30 seconds, and then t r a n s f e r r e d to isopentane f o r 10 seconds, and d r i e d by t o u c h i n g t o f i l t e r paper.  finally  The s t a i n was made as  f o l l o w s : 10 u l of c o n c e n t r a t e d HC1 were added t o 250 mg of u r a n y l a c e t a t e , a f t e r which 10 ml of d i s t i l l e d water were added (UA s t o c k ) .  For use, 10 u l of UA stock were d i l u t e d w i t h 10  ml of 70% e t h a n o l . only.  G r i d s may  The d i l u t e d s t a i n i s s t a b l e f o r one hour  be observed without shadowing  or may  shadowed w i t h p l a t i n u m at an angle of 5 to 7°. i n c r e a s e s c o n t r a s t and g r i d s t a b i l i t y  be  Shadowing  (from one day to one week).  G r i d s were observed and photographed i n a P h i l i p s EM300 e l e c t r o n microscope a t m a g n i f i c a t i o n s of 10,000 t o 40,000 t i m e s . photography s e s s i o n a c a l i b r a t i o n g r i d mm)  (E.F. F u l l a m , 2160  was photographed a t each m a g n i f i c a t i o n used.  measured on photographic enlargements u s i n g a map  DNA  At each lines/  lengths were  measuring d e v i c e  (Dietzgen) . R a d i o i s o t o p e l a b e l l i n g of UGV-nucleic a c i d JJ. g i g a s c u l t u r e s were grown f o r two weeks i n B e i j e r i n c k ' s medium w i t h t r i t i u m - l a b e l l e d u r a c i l or thymidine added i n o r d e r t o produce l a b e l l e d VLP n u c l e i c a c i d . ml seeder t e s t tube was  One  10  added to 100 ml of f r e s h u n l a b e l l e d  medium, shaken, and then 10 ml was dispensed t o each of 11 t e s t tubes.  The new  c u l t u r e s were heat-shocked a t 38 C i n the dark  for  6 hours.  activity  Then 0.2 ml of  H-uracil  (1 mCi/ml, s p e c i f i c  of 20 Ci/mmol, Amersham/Searle; f i n a l c o n c e n t r a t i o n  i n medium of 20 uCi/ml) was  added to each of two tubes w i t h  0.1 ml of 10 micromolar u n l a b e l l e d u r a c i l .  To two other  3 tubes 0.10. ml of  H-thymidine  (0.5 mCi/ml, sp. a c t . 23 Ci/mmol,  Amersham/Searle; f i n a l c o n c e n t r a t i o n i n medium of 5 was  added w i t h 0.1 ml of u n l a b e l l e d 10 y-M  technique was Two  used  thymidine.  samples  of 0.1 ml each were withdrawn from each  f o r the f o u r t e e n days f o l l o w i n g .  added and every other day  The 0.1  samples were each  added t o 3 ml of i c e c o l d 5% t r i c h l o r o a c e t i c a c i d  was  (BSA,  1 mg/ml, 0.1 ml) was  l e f t on i c e f o r 5 t o 10 minutes.  m a t e r i a l was  The  (TCA).  Bovine  added, and the mixture acid-insoluble  c o l l e c t e d by f i l t r a t i o n through a 0.45  membrane, which was  Sterile  throughout.  tube immediately a f t e r the l a b e l was  serum albumin  uCi/ml)  y Millipore  then washed 3 times with c o l d 5% TCA,  w i t h c o l d 95% e t h a n o l , and then d r i e d at 70 C.  twice  The f i l t e r  was  then put i n a s c i n t i l l a t i o n v i a l c o n t a i n i n g 10 ml of Aquasol (New  England N u c l e a r ) .  counter was  A P i c k e r Nuclear Liquimat s c i n t i l l a t i o n  used; samples were e q u i l i b r a t e d at 4 C i n the dark  overnight before counting.  The c o u n t i n g e f f i c i e n c y f o r the  t r i t i u m - l a b e l l e d n u c l e o s i d e s was uracil  determined to be 74% f o r the  and 56% f o r the thymidine by adding known volumes of  pure l a b e l d i r e c t l y to s c i n t i l l a n t and comparing to  counts expected from the s p e c i f i c At  a c t u a l counts  activities.  the end of the growth p e r i o d , 0.1 ml "of the  remaining c u l t u r e medium was  p l a t e d onto n u t r i e n t agar t o t e s t  f o r b a c t e r i a l contamination; the remaining medium  (about 8 ml)  was passed through a 0.45 u M i l l i p o r e f i l t e r .  The f i l t e r e d  c u l t u r e medium was c e n t r i f u g e d a t 9,000 rpm f o r one hour w i t h 0.2 ml of u n l a b e l l e d VLP's and loaded onto an SW 41 g r a d i e n t at  pH 8.0 (10 t o 40% sucrose i n 0.05 M T r i s B u f f e r pH 8.0)  which was then c e n t r i f u g e d a t 10,000 rpm f o r 30 minutes i n the Beckman L5-75 u l t r a c e n t r i f u g e . at  Resolved g r a d i e n t s were scanned  254 nm i n an ISCO Model UA 5 absorbance monitor;, ten-1.2 ml  f r a c t i o n s were c o l l e c t e d from each g r a d i e n t .  Each f r a c t i o n was  d i l u t e d w i t h 2 ml of i c e c o l d 10% TCA, incubated a t 4 C f o r 30 minutes, and f i l t e r e d through Whatman 3 MM paper p r e v i o u s l y soaked i n 5% sodium pyrophosphate and d r i e d .  The f i l t e r s were  batch r i n s e d i n 5% TCA f o r 30 minutes; the r i n s i n g was repeated twice.  The f i l t e r s were then r i n s e d 2 t o 3 times w i t h c o l d  e t h a n o l and f i n a l l y w i t h one change of ether and a i r d r i e d . The experiment was repeated w i t h both the thymidine and u r a c i l l a b e l s s u p p l i e d a t 10 uCi/ml and h a r v e s t of a l g a l c e l l s a f t e r one week o f growth.  Amicon 0.45 u f i l t e r s  s u b s t i t u t e d f o r both the M i l l i p o r e f i l t e r s  and the Whatman 3 MM  paper a f t e r comparisons r e v e a l e d no d i f f e r e n c e s i n r e t e n t i o n characteristics. Heat shock experiments A growth experiment was performed twice t o t e s t f o r heat-induced r e l e a s e of VLP's.  In the f i r s t experiment one-10 ml  seeder t e s t tube o f mature U. g i g a s f i l a m e n t s was added t o each of  twelve-125 ml f l a s k s c o n t a i n i n g 50 ml of B e i j e r i n c k ' s medium.  Immediately a f t e r seeding, e i g h t of the f l a s k s were h e l d a t 39 C for  6 hours w i t h normal l i g h t i n g ? - four were maintained a t the  normal temperature of approximately 22 C.  A f t e r heat  treatment the f l a s k s were t r a n s f e r r e d t o a r o t a r y shaker a t low speed f o r two weeks of growth.  Then the contents of each f l a s k  were i n d i v i d u a l l y f i l t e r e d through Whatman #1 f i l t e r paper; the c u l t u r e medium was  centrifuged  the r e s u l t i n g p e l l e t was pH 7.25.  at 9,0 00 rpm  f o r one hour, and  taken up i n 2 ml of 0.05  Numbers of VLP's r e l e a s e d  by the e l e c t r o n m i c r o s c o p i c a l  M  Tris-HCl  i n each f l a s k was  c o u n t i n g procedure  determined  previously  described. In the second experiment, zoospores were produced the n i g h t b e f o r e seeding by adding ten-10 ml seeder tubes to 400 of f r e s h medium; i n the morning medium was  ml  10 ml of z o o s p o r e - c o n t a i n i n g  added to each of 24-125 ml f l a s k s .  Twelve f l a s k s  were then heat^-shocked a t 38 C f o r seven hours i n the dark, during  what would normally be the c u l t u r e ' s p h o t o p e r i o d .  twelve c o n t r o l s had the normal day-time temperature and regime.  light  Assay methods f o r VLP's a t the end of two weeks were  i d e n t i c a l t o experiment 1.  A l g a l c e l l s were c o l l e c t e d by  f i l t r a t i o n and pooled i n two groups, heat-shocked and for  The  assay of c h l o r o p h y l l  a. content.  In l a t e r c u l t u r i n g of JJ. g i g a s ,  the heat shock at 3 8 C  i n the dark, f o r 6 to 8 hours of the p h o t o p e r i o d , was used to promote r e l e a s e  controls,  of VLP's.  routinely  -44-  RESULTS I.  I n t e r a c t i o n of UGV A.  w i t h Uronema gigas  Morphology of UGV  and  i t s host a l g a  Uronema g i g a s , Indiana U n i v e r s i t y C u l t u r e C o l l e c t i o n number 17 4, was  obtained i n 197 4 by Dr. J . A l l a n Dodds.  E l e c t r o n m i c r o s c o p i c examination  of s u b - c u l t u r e d c e l l s  the presence of v i r u s - l i k e p a r t i c l e s  (VLP's) i n the cytoplasm  a few c e l l s , as d e s c r i b e d by Mattox, Stewart, and F l o y d Negative was  confirmed of  (1972).  s t a i n i n g of a l i q u o t s of the medium i n which the a l g a  grown r e v e a l e d t h a t s i m i l a r VLP's had been r e l e a s e d from the  algal cells.  F i g u r e s l a and  l b show a h e a l t h y f i l a m e n t i n  l o n g i t u d i n a l and c r o s s s e c t i o n s . n u c l e o l u s and  A d i s t i n c t nucleus  i n t a c t n u c l e a r membrane can be seen;  ribosomes, and  with  mitochondria,  the l a r g e g i r d l i n g c h l o r o p l a s t c o n t a i n i n g a  p y r e n o i d are a l s o p r e s e n t .  The c e l l s are bound by a u n i t membrane  and a r i g i d c e l l w a l l ; plasmodesmata between neighboring be seen i n F i g u r e l a . relatively  cells  can  F i g u r e s l c , d, and e show three c e l l s at  l a t e stages of i n f e c t i o n when c e l l u l a r membrane  systems have become completely r e f e r r e d to as UGV, the cytoplasm; degeneration  disorganized.  VLP's  (also  f o r convenience) are s c a t t e r e d throughout  no n u c l e a r membrane can be d e t e c t e d ,  of c y t o p l a s m i c  and  and c h l o r o p l a s t membrane systems i s  f a r advanced. The VLP's are unusual  i n morphology and  shown i n F i g u r e 2b, approximately  size.  As  10% of the r e l e a s e d p a r t i c l e s  have t a i l s which vary i n l e n g t h to a maximum of approximately  one  -45-  Figure  1.  T h i n s e c t i o n s of h e a l t h y and i n f e c t e d c e l l s of Uronema g i g a s . T i s s u e was f i x e d i n g l u t a r a l d e h y d e and osmium t e t r o x i d e , embedded i n Epon, and s t a i n e d with u r a n y l a c e t a t e and lead c i t r a t e . a) Healthy f i l a m e n t showing s e v e r a l i n t a c t c e l l s . b) C r o s s - s e c t i o n of h e a l t h y c e l l . c) C r o s s - s e c t i o n of c e l l c o n t a i n i n g VLP's. d) Two-celled germling with VLP's. . e) C r o s s - s e c t i o n of c e l l c o n t a i n i n g VLP's.  -46-  Figure  2.  P u r i f i e d p a r t i c l e s o f UGV i n n e g a t i v e s t a i n (5% p h o s p h o t u n g s t i c a c i d pH 7,2) a) VLP's w i t h d e n s e b o d i e s . b) V L P ' s a f t e r p u r i f i c a t i o n t o s o l u b i l i z e d e n s e bodies. c) V L P ' s w i t h p a r t i c l e s o f T I V and TMV f o r s i z e comparisons.  -47-  F i g u r e 3.  a) High m a g n i f i c a t i o n photograph of a VLP w i t h i n an a l g a l c e l l . Note membrane-like coat, nonhomogeneous c o r e . b) High m a g n i f i c a t i o n photograph of a p u r i f i e d VLP, without t a i l , i n negative s t a i n . c) T a i l e d VLP's w i t h i n degenerating c e l l s of JJ. g i g a s . d) Higher m a g n i f i c a t i o n of ( c ) . e) An e a r l y stage of i n f e c t i o n of VLP's. Note i n t a c t c h l o r o p l a s t and a n g u l a r i t y of VLP coats  -'4 8m i c r o n , and which o f t e n e x h i b i t a at  about h a l f t h e i r l e n g t h .  c e l l s , the UGV  knob o r round  protruberance  Seen i n c r o s s - s e c t i o n s w i t h i n the  head o r c a p s i d appears t o be i c o s a h e d r a l ;  p e n t a g o n a l and hexagonal  forms are b o t h seen i n the VLP's  shown i n F i g u r e 1, s u g g e s t i n g the v a r i o u s p l a n e s of c u t t i n g an i c o s a h e d r o n , as d e s c r i b e d by Home (1974).  The VLP's v a r y  i n the f u l l n e s s of the c a p s i d ; o f t e n a c a p s i d appears t o have a c e n t r a l empty space, w i t h a s t r o n g l y o s m i o p h i l i c a r e a next to  the VLP c o a t and d e l i c a t e f i b e r s f i b e r s  throughout  the c e n t r a l a r e a .  radiating  The o u t e r s h e l l of the UGV  particle  i s a l s o o f i n t e r e s t : i t appears t o c o n s i s t of a membranous l a y e r , somewhat r e s e m b l i n g a c e l l u l a r plasmalemma, however i t a l s o shows a c e r t a i n r i g i d i t y i n shaping i n t o the hexagonal seen i n s e c t i o n s (e.g. F i g u r e 3 a ) .  forms  F i v e o r s i x rounded v e r t i c e s ,  s t r o n g l y n e g a t i v e l y s t a i n i n g , were o c c a s i o n a l l y seen, embedded i n o r a t t a c h e d t o the o u t e r s h e l l the VLP's was  ( F i g u r e 3b).  The d i a m e t e r of  a p p r o x i m a t e l y 390 nm when p a r t i c l e s were f i x e d i n  g l u t a r a l d e h y d e b e f o r e n e g a t i v e s t a i n i n g o r embedding; VLP d i a m e t e r ranged up t o 590 nm under v a r y i n g c o n d i t i o n s of pH stain  and  (see Table I X ) . B.  Cytopatholoqy  V i r u s - l i k e p a r t i c l e s were u s u a l l y found i n c e l l s a t advanced s t a g e s o f d e g e n e r a t i o n , w i t h l i t t l e o r g a n i z a t i o n of membranes l e f t .  An i n t a c t , normal n u c l e u s , as seen i n F i g u r e s  l a and l b , was  u s u a l l y p r e s e n t i n s e c t i o n s of h e a l t h y a l g a l  c e l l s , but was  never observed  i n an i n f e c t e d c e l l , even a t  e a r l y s t a g e s of VLP m a t u r a t i o n .  The r o l e of the n u c l e u s i n e a r l y  "49-  events of UGV  r e p l i c a t i o n i s not c l e a r however, s i n c e the  breakdown of the n u c l e a r membrane i s f o l l o w e d by d i s i n t e g r a t i o n of  a l l the major c e l l u l a r membrane systems, i n c l u d i n g the  l a m e l l a e of the c h l o r o p l a s t , at l a t e r stages of UGV  infection.  A very e a r l y stage of i n f e c t i o n i s shown i n F i g u r e 3e.  The p h o t o s y n t h e t i c l a m e l l a e appear normal.  c l e a r demarcation of the nucleoplasm.  There i s no  Outer s h e l l s of the  UGV  p a r t i c l e can be seen forming i n p i e c e s with the a n g u l a r i t y of completed p o l y h e d r a , e n c l o s i n g some ground substance which appears almost i d e n t i c a l to the surrounding m a t e r i a l .  A  h a l o - l i k e , c l e a r area i s sometimes apparent around the o u t e r edge of the d e v e l o p i n g p a r t i c l e s . T a i l s were o c c a s i o n a l l y observed on d e v e l o p i n g UGV p a r t i c l e s w i t h JJ. g i g a s c e l l s of  (Figures 3c and d) .  c a p s i d assembly, p a c k i n g , and t a i l  attachment  The  sequence  i s not c l e a r  from the few c e l l s observed i n s e c t i o n a t i n t e r m e d i a t e stages of  UGV  development. V i r u s - l i k e p a r t i c l e s were observed i n a l g a l germling  c e l l s o n l y ; 'none of the zoospores or expanded c e l l s of f i l a m e n t s which were s e c t i o n e d ever showed s i g n s of i n f e c t i o n . It  i s p o s s i b l e t h a t the s e t t l i n g germling i s the most s u s c e p t i b l e  stage i n the l i f e c y c l e of JJ. g i g a s f o r VLP r e l e a s e and t h a t i n d i v i d u a l germlings which become i n f e c t e d a t t h i s stage f a i l to  develop f u r t h e r . Examination of t h i n s e c t i o n s of heat-shocked  p o p u l a t i o n s of germlings r e v e a l e d c e l l s c o n t a i n i n g VLP's a t about f i v e times the frequency of scans of mixtures of c e l l s at  a l l stages i n the l i f e  c y c l e of the a l g a , prepared without  -50heat shock.  About one out of twenty heat-shocked  germlings  t h a t were s e c t i o n e d c o n t a i n e d VLP's a t some stage of C.  assembly.  Scanning e l e c t r o n microscopy of germlings  Newly r e l e a s e d zoospores were allowed to s e t t l e  on  d i a l y s i s membranes a t the s u r f a c e of the c u l t u r e medium i n s m a l l beakers.  A f t e r one to two days of growth,  the membranes  were c o l l e c t e d , f i x e d , and then c r i t i c a l - p o i n t - d r i e d f o r examination by scanning e l e c t r o n microscopy. or treatments w i t h heavy metal s t a i n s was  No metal c o a t i n g  done i n order t o a l l o w  use of the x-ray m i c r o a n a l y s i s u n i t f o r c h a r a c t e r i z a t i o n of the composition of the a l g a l  cells.  A r e p r e s e n t a t i v e f i e l d of the young germlings i s shown i n F i g u r e 4a.  Little  s t r u c t u r e i s r e v e a l e d beyond the  g e n e r a l o u t l i n e of the c e l l s ; one type of f i x a t i o n  caused  c o l l a p s e of the c h l o r o p l a s t i n t o an elongated c u p - l i k e At h i g h e r m a g n i f i c a t i o n s , about 5% of the germlings  form.  appeared  to c o n t a i n m u l t i p l e , opaque spheres of roughly 500 nm  diameter  which could be seen through the c e l l w a l l and a f t e r c r u s h i n g to p a r t i a l l y r e l e a s e the p a r t i c l e separately.  (Figure 4 c ) , c o u l d be analysed  Elemental x-ray m i c r o a n a l y s e s c o n s i s t e n t l y  the presence of more phosphorus a l g a l cytoplasm; potassium was  revealed  i n the spheres as compared to somewhat i n c r e a s e d as w e l l .  Figure  4d shows r e p r e s e n t a t i v e a n a l y s i s of cytoplasm versus the dense spheres; the l a r g e peak occurs at 2.02 phosphorus  secondary e l e c t r o n s .  t h e i r elemental phosphorus  KeV,  the energy l e v e l of  The diameter of the spheres and  content suggest t h a t they may  p a r t i c l e s , f i x e d in. s i t u w i t h i n i n f e c t e d c e l l s .  be  UGV  Potassium  and  sodium occur with n u c l e i c a c i d s as c o u n t e r - i o n s ; the f i x a t i o n i n potassium potassium. x-ray.  g l u t a r a l d e h y d e would r e p l a c e any sodium ions w i t h An attempt was made t o analyse p u r i f i e d VLP's by  P u r i f i e d VLP's, d r i e d down d i r e c t l y onto a scanning  e l e c t r o n microscope stub, c o u l d not be seen--the s t r u c t u r e was l o s t - - b u t x-ray a n a l y s i s i n d i c a t e d the presence  o f phosphorus,  s u l f u r , sodium, and c a l c i u m , when many a p p l i c a t i o n s of a s o l u t i o n o f VLP's were a p p l i e d t o a carbon  stub.  S u l f u r and  c a l c i u m may be d i f f i c u l t t o d e t e c t i n the minute q u a n t i t i e s p r e s e n t i n a s i n g l e p a r t i c l e , f i x e d i n s i t u i n the a l g a l  cell.  -52-  Figure  4.  S c a n n i n g e l e c t r o n m i c r o s c o p y and x - r a y m i c r o a n a l y s i s o f JJ. g i g a s g e r m l i n g s . a) A l g a l g e r m l i n g s on d i a l y s i s membranes. b) C l o s e - u p o f g e r m l i n g t i p c o n t a i n i n g opaque s p h e r e s . c) C r u s h e d g e r m l i n g w i t h s p h e r e made a c c e s s i b l e f o r x-ray m i c r o a n a l y s i s . d) E l e m e n t a l a n a l y s i s o f d e n s e s p h e r e ( l o w e r c u r v e ) compared t o a l g a l c y t o p l a s m (upper c u r v e ) . Large peak on l e f t i s a t t h e e n e r g y l e v e l o f p h o s p h o r u s ; t h e s m a l l e r peak on t h e r i g h t r e p r e s e n t s p o t a s s i u m .  S 9.0U  -53-  5n  Figure  5.  Growth of U. g igas and r e l e a s e o f V L P ' s in two c u l t u r e media: B e i j e r i n c k ' s medium ( B J ) , and Br i s t o l ' s med ium (BR).  -54II.  Isolation A. JJ.  mineral  o f UGV  Growth o f t h e host  alga  gigas  was grown i n l a r g e v o l u m e s o f B e i j e r i n c k ' s  medium f o r p u r i f i c a t i o n  environmental exposed to  and p u r i f i c a t i o n  conditions  t o approximately  5000 l u x d u r i n g  J.A. Dodds).  richer that  not  (Bristol's  r e l e a s e were g r e a t e r  shaker,  which  after  (results of  medium, S t e i n 1 9 7 3 ) ,  5.  algal  revealed  Results are  o f c u l t u r e d U. g i g a s  t h e a l g a was grown i n s m a l l shortened  a  growth and  i n B e i j e r i n c k ' s medium.  When l a r g e v o l u m e s  a t 20  about two  a d d i t i o n o f f r e s h medium  i n t h e two month growth p e r i o d , b o t h  essential,  cultures,  C o m p a r i s o n o f B e i j e r i n c k ' s medium w i t h  g r o w t h medium,  shown i n F i g u r e  Under t h e  the photoperiod,  23 C, m a x i m a l v i r u s r e l e a s e w a s o b s e r v e d  Dr.  VLP's.  available--non-shaking  months o f growth, w i t h o u t  VLP  of liberated  t h e growth period  flasks  were  on a r o t a r y  t o approximately  one  month. Without counting relied culture The  procedure  and over  VLP  assay,  estimation  the course  techniques  of particles  1972, Sharp  concentration  a purification,  1975).  h a d t o be  o f VLP c o n c e n t r a t i o n p e r  of various  f o r accurate  the electron microscope  o f a "VLP i n d e x " )  m e t h o d u s e d was r e l a t i v e l y  determinations Meyers  (determination  upon f o r a c c u r a t e  counting  droplet  an i n f e c t i v i t y  purification crude  procedures.  compared  t o spray  electron microscopical  p e r u n i t volume  (Haschemeyer and  However, t h e number o f a s s a y s o f  t o be done, e s p e c i a l l y t h r o u g h  d i c t a t e d the use of a simpler  the steps of  method.  Some  -55verification counting as  the  of  the  a dilution  algal  drawback of  was  poor binding  of  detergents.  could  not  by  U_.  gigas  in  terms of  o f VLP of  index  per  only Dr.  High v i r u s - y i e l d i n g  was  not  B.  sense w i t h  of  the  49  the  as  in  terms  milligram  (Table  V).  a decline  in  subcultures  isolates  a wide range of  Extensive a  and  absolutely correlated  subcultures  was  of  by  had  lost  rates  of  the  were s e l e c t e d f o r  algal  testing  that  to ensure  stable characteristic  of  each  performed.  Purification  Initial h a m p e r e d by  be  none of  for virus purification.  detergent  highly variable  liberated  Dodds i s o l a t e d  was  presence  harvests  c u l t u r e medium  i n a broad  to release VLP's, there  liberation  r e l e a s e was  not  One  concentration  of  growth, expressed  could  well  IV.  i n the  from repeated  of  by  concentration.  particles  algal  VLP  i n v o l v i n g use  and  transfer; while  r a t e o f VLP  isolate  and  obtained  i n Table  surface  f o r VLP  millilitre  a content.  zoospore  release. growth  steps  synthesis  u n i t of  was  determining  a grid  accurately  VLP's r e l e a s e d  chlorophyll  capacity  method of  a c t u a l numbers of  c u l t u r e age,  single  presented  of VLP's t o  t h a t VLP  technique  T i p u l a i r i d e s c e n t v i r u s as are  Purification  c h l o r o p h y l l a per  with  1.  this  our  consistent observation  was  Numbers o f  the  Results  assayed  A  of  s e r i e s of  VLP's.  further the  accuracy  attempts  VLP's  to purify  the  VLP  from JJ. g i g a s  were  four factors:  the  presence  one  fifth  in  density  the  i n the  g r o w t h medium o f  diameter  of  the  (shown i n F i g u r e  VLP, 2a);  spheres  but  approximately  identical  to the  VLP  "56" TA'BLE IV -- E l e c t r o n microscope counting technique f o r assay of VLP c o n c e n t r a t i o n C o n c e n t r a t i o n (ug/ml) of d i l u t i o n of preparation TIV  6.9 3.5 1.8  UGV  1:5 1:10 1:50 1:100  P a r t i c l e s per transect 3  Particle dilution  102 43 25  102 86 100  70 39 6.6 3.2  350 390 330 320  Average of p a r t i c l e s counted i n 10 g r i d square t r a n s e c t s . TABLE V —  Release of UGV p a r t i c l e s i n r e l a t i o n t o a l g a l growth, measured by c h l o r o p h y l l a. content per ml of c u l t u r e medium. 3  Age of culture (weeks)  Chlorophyll a content (mg/ml)  VLP index  VLP index c h i a_ (mg  9  2.7  1100  407  8  3.2  392  123  8  2.8  244  87  10  1.1  70  64  8  3.2  200  63  10  2.8  150  54  7  2.1  112  53  9  3.2  165  52  8  3.3  70  21  8  1.9  40  21  6  5.5  37  7  8  3.7  16  4  6  4.6  8  2  Data from t h i r t e e n r e p r e s e n t a t i v e  harvests.  -572.  the low c o n c e n t r a t i o n s of VLP's i n l a r g e volumes of c u l t u r e medium;  3.  the tendency of the VLP's to aggregate i r r e v e r s i b l y a t pH 5.5  or below, when present  a purification; 4.  i n high c o n c e n t r a t i o n s  during  and  the presence of a l g a l c e l l u l a r d e b r i s i n the medium  (even  though no homogenization of a l g a l c e l l s had been done). Methods assayed Table VI.  to circumvent  these problems are d e t a i l e d i n  T h i s t a b l e d e s c r i b e s r e s u l t s of v a r i o u s p u r i f i c a t i o n s  i n terms of VLP  q u a l i t y r a t h e r than q u a n t i t y s i n c e , as  p r e v i o u s l y mentioned, h a r v e s t s of U. gigas v a r i e d widely u n p r e d i c t a b l y i n numbers of VLP's r e l e a s e d . of  and  D i r e c t comparisons  y i e l d s of VLP's from h a r v e s t to h a r v e s t would not  be  meaningful as a measure of the e f f i c i e n c y of a p a r t i c u l a r p u r i f i c a t i o n procedure. presented  The  f i n a l procedure developed  is  i n F i g u r e 6 with a r e p r e s e n t a t i v e example of the  r e l a t i v e amounts of VLP's at each stage of p u r i f i c a t i o n . A l g a l f i l a m e n t s were f i r s t removed from the c u l t u r e medium by f i l t r a t i o n or paper f i l t r a t i o n for  determinations  through b o l t i n g s i l k , to remove zoospores.  C e l l s were r e s e r v e d  of c h l o r o p h y l l aj the f i l t r a t e  c e n t r i f u g e d at 9,000 rpm b o d i e s , and  f o l l o w e d by membrane  f o r one  was  hour to p e l l e t VLP's, dense  some d e b r i s .  The dense bodies were p u r i f i e d by two  c y c l e s of  d i f f e r e n t i a l c e n t r i f u g a t i o n of a h a r v e s t which contained few VLP's.  E n e r g y - d i s p e r s i v e spectroscopy  b o d i e s , k i n d l y performed by Mr.  very  of the dense  George J . Georgakopoulos of the  Department of Geology, U.B.C., demonstrated t h a t i r o n ,  calcium,  -58TABLE V I -- Comparison o f c o n c e n t r a t i o n and p u r i f i c a t i o n methods f o r UGV as determined by e l e c t r o n m i c r o s c o p y o f ' preparations. Method assayed  Aggregated particles  Dense bodies  F i l t r a t i o n , then 10,000 x g f o r one hour  Cellular debris  Loss o f particles  +  +  Sucrose c u s h i o n f o r + c o n c e n t r a t i o n and further p u r i f i c a t i o n  +  45-90% s u c r o s e density gradient centrifugation  +  ±  Polyethylene g l y c o l (PEG) p r e c i p i t a t i o n df V L P s from medium 4% PEG and 0.25 M NaCl 4% PEG, no NaCl  +  1  PEG/sucrose i n v e r s e g r a d i e n t s : 10% s u c r o s e , 10% PEG t o 40% s u c r o s e , 2.5% PEG  No V L P s were precipitated, with or without NaCl. 1  +  Na a c e t a t e b u f f e r , pH ± 5.5 t o resuspend VLP pellet after centrifugation To r e v e r s e a g g r e g a t i o n of VLP's: added 0.01 M EDTA N/A added 0.03 M MgCl N/A sonication N/A added Igepon T-73, N/A a detergent  c  No change No change No change  0.01 M EDTA added t o resuspension b u f f e r , pH 5.5  (made s o l u b l e )  Sodium c i t r a t e o r Sodium a c e t a t e b u f f e r , pH 6.0 f o r resuspension of VLP' s  (made s o l u b l e )  +  + + +  N/A N/A N/A N/A  -59*  .Method assayed  Dense bodies  Aggregated particles  Cellular debris  0.05 M sodium citrate/ 0.01 M EDTA pH 6.0 (made s o l u b l e ) for resuspension of VLP's  +  Ammonium s u l f a t e p r e c i p i t a t i o n of VLP's (assayed 15 t o 55%)  +  +  Potassium t a r t r a t e / glycerol gradient centrifugation ( O b i j e s k i e t a l . 1974) T r i t o n X-100 d e t e r gent added a t 0.5% with citrate/EDTA buffer  Loss o f particles  +  -  Very few lost.  not  applicable.  e l e c t r o n m i c r o s c o p e assay was u n r e l i a b l e i n t h e presence o f detergent.  -60-  PURIFICATIQN OF UGV  Relative yields: 100 % |  Filtr ation  F  f iltVatc  cells  I T  chlorophyll a assay  Centrifuga fug at ion 10,000 g 1 hour ;  pellet JE  supernatant (discard)  +  resuspend in Citrate! EDTA! Triton buffer  I  r  dialysis ^ Centrifugation  74%  1  supernatant (discard)  pellet  J  resuspend in Tris-HCI buffer i 56% dialysis  I  Sucrose density gradient centrifugation 10 000 g 30 minutes ;  I collect band of V L P ' s dialysis  42%  •  store in Tris-HCI F i g u r e 6.  F i n a l p u r i f i c a t i o n procedure f o r UGV p a r t i c l e s . d e t a i l s see M a t e r i a l s and Methods.  For  p o t a s s i u m , manganese, and copper were p r e s e n t . not q u a n t i t a t i v e .  The t e c h n i q u e i s  I t proved p o s s i b l e t o remove the dense b o d i e s  by c h e l a t i o n w i t h EDTA a t a pH below 6.0; however, e x t e n s i v e a g g r e g a t i o n of VLP's r e s u l t e d i f the c h e l a t i o n s t e p was p r o l o n g e d , e.g. over 48 h o u r s .  A l g a l d e b r i s was removed d u r i n g  the c h e l a t i o n s t e p by the a d d i t i o n of 0.5% T r i t o n d e t e r g e n t t o the E D T A - c o n t a i n i n g b u f f e r .  X-100  A f t e r a second  c e n t r i f u g a t i o n , the VLP's were resuspended i n a 0.05  M Tris-HCl  b u f f e r a t pH 8.0 t o l i m i t a g g r e g a t i o n of the VLP's.  Loss of  VLP's over the c o u r s e o f a p u r i f i c a t i o n was due m a i n l y t o a g g r e g a t i o n and consequent p e l l e t i n g i n c e n t r i f u g a t i o n s t e p s . When adequate amounts of VLP's were a v a i l a b l e  after  the d i a l y s i s s t e p of p u r i f i c a t i o n , the p a r t i c l e s were f u r t h e r p u r i f i e d by d e n s i t y g r a d i e n t c e n t r i f u g a t i o n on 10-40% o r 20-50% s u c r o s e g r a d i e n t s a t pH 8.0.  A r e p r e s e n t a t i v e scan of r e s o l v e d  g r a d i e n t s i s shown i n F i g u r e 7a.  Suspensions of TIV were always  c e n t r i f u g e d i n one g r a d i e n t of each run as a s t a n d a r d . Comparing the two c u r v e s , i t i s i m m e d i a t e l y apparent t h a t the VLP from JJ. g i g a s i s much l e s s homogeneous i n the p r o p e r t i e s which a f f e c t s e d i m e n t a t i o n than i s TIV.  Bands from the g r a d i e n t s  of UGV were always b r o a d , o c c a s i o n a l l y r e s o l v i n g i n t o s e p a r a t e a r e a s , as shown i n F i g u r e 7b.  two  By e l e c t r o n m i c r o s c o p y ,  the upper band appeared t o c o n t a i n p a r t i a l l y empty VLP  capsids,  and some p a r t i a l l y degraded p a r t i c l e s ; the lower band c o n t a i n e d complete p a r t i c l e s which l o o k e d homogeneous i n morphology. T a i l e d p a r t i c l e s were d i s t r i b u t e d i n b o t h bands.  I n some runs  a f a s t e r s e d i m e n t i n g band c o n t a i n i n g aggregates o f the VLP's were d e t e c t e d .  The r e l a t i o n s h i p between s e d i m e n t a t i o n d e p t h ,  -62A.  0.01 sodium acetate pH5.5 12,000 g x 26 min  B. 0.05  M Tris-HCI  10,000 g x I. empty VLP's II. incomplete V L P ' s , tails III-intact V L P ' s , tails  Bottom  F i g u r e 7-  Centrifugation  o f UGtf p a r t i c l e s  i n 10-40% s u c r o s e  gradients.  -6 3-  UV absorbance, and p a r t i c l e counts of UGV 7a.  i s shown i n F i g u r e  The area of the g r a d i e n t with' the h i g h e s t absorbance a t  254 nm a l s o c o n t a i n e d the g r e a t e s t number of p a r t i c l e s by e l e c t r o n microscopy. A l g a l c e l l d i s r u p t i o n was  attempted by v a r i o u s methods  d e s c r i b e d i n Table VII t o l i b e r a t e i n c r e a s e d amounts of VLP's. A l g a l c e l l s were d i f f i c u l t to homogenize; most methods were only partially effective.  The only procedure which r e s u l t e d i n  d i s r u p t e d c e l l s , s o n i c a t i o n f o l l o w e d by f r e e z i n g and showed no i n c r e a s e d y i e l d of VLP's.  thawing,  Because of t h i s r e s u l t ,  no  f u r t h e r attempts were made at homogenization. C.  Heat shock e f f e c t s on VLP  yield  The r e s u l t s of the two heat shock experiments are shown i n Table V I I I . shock was  In the f i r s t experiment, i n which heat  a p p l i e d d u r i n g the normal p h o t o p e r i o d , no  s i g n i f i c a n t i n c r e a s e i n the number of r e l e a s e d VLP's was observed between heat-shocked and c o n t r o l f l a s k s , although the number o f VLP's r e l e a s e d per heat-shocked f l a s k had a h i g h e r mean.  The wide v a r i a b i l i t y of numbers of VLP's r e l e a s e d among  each treatment could-have been due t o the d i f f e r e n c e s i n m a t u r i t y of the seeding c e l l s s u p p l i e d ; i n t h i s experiment a mixture of mature f i l a m e n t s , young germlings, and zoospores  was  used t o begin the c u l t u r e . In the second experiment the heat shock  was  administered i n darkness d u r i n g what would have been a normal photoperiod.  There were approximately s i x times as many VLP's  r e l e a s e d from heat-shocked f l a s k s ' as from the c o n t r o l s , comparing  the means.  C o n t r o l f l a s k s had an average c h l o r o p h y l l e  -64-  TABLE V I I —  C e l l d i s r u p t i o n methods assayed f o r U. g i g a s  Method  Cellular disruption  G r i n d i n g w i t h sand Grinding with l i q u i d nitrogen Homogenization w i t h Waring b l e n d o r Sonication of c e l l s S o n i c a t i o n f o l l o w e d by f r e e z i n g and thawing  TABLE V I I I —  R e l e a s e o f VLP's  +  Heat-shock e f f e c t s on UGV r e l e a s e Experiment 1 L i g h t a t 38 C  Experiment 2 Dark a t 38 C Chi a content  Controls  41±50 (4)  36±5  Heat-shocked  98193  a  a  (8)  b  (12)  228180(12)°  3.2 mg/ml 2.5 mg/ml  F i g u r e s a r e g i v e n i n terms o f "VLP i n d e x " : a c t u a l numbers o f VLP's counted c o n v e r t e d t o a 100 X c o n c e n t r a t i o n o f t h e o r i g i n a l volume o f c u l t u r e medium. The means o f each group, p l u s o r minus one s t a n d a r d d e v i a t i o n , a r e g i v e n , F i g u r e s i n p a r e n t h e s e s r e f e r t o numbers o f f l a s k s assayed. Heat shock was a d m i n i s t e r e d i n t h e dark d u r i n g what would be a normal p h o t o p e r i o d .  -6 5content of 3.2 mg/ml; heat-shocked f l a s k s averaged 2.5 mg/ml of c h l o r o p h y l l a_. Both the c o n t r o l s and the heat-shocked c u l t u r e s showed l e s s v a r i a b i l i t y i n VLP numbers r e l e a s e d than i n experiment one, perhaps because these c u l t u r e s were begun w i t h a uniform suspension of f r e s h l y r e l e a s e d  zoospores  only.  In l a t e r c u l t u r i n g of JJ_. g i g a s f o r v i r u s p u r i f i c a t i o n , a s i x t o e i g h t hour heat shock i n darkness was i n c l u d e d a f t e r zoospore r e l e a s e , based on the i n c r e a s e observed i n the second  experiment.  i n y i e l d of VLP's  -66III.  P a r t i a l characterization A.  Dimensions of  of UGV  and  UGV  Data f o r measurements of UGV IX.  i t s components  are presented i n Table  P a r t i c l e s were measured in. s i t u i n i n f e c t e d  vitro after purification. variety  and  of c o n d i t i o n s of s t a i n and  pH,  (UA)  Aside from the  and  data  phosphotungstic  acid  s t a i n s were used, u r a n y l formate, potassium molybdate,  potassium permanganate s t a i n s were a l s o t r i e d , at v a r i o u s  E f f e c t s on VLP structure  dimensions were s i m i l a r and  of the VLP's was  not  resolution  of  hyde (and staining  o f t e n osmium t e t r o x i d e or embedding and  diameter of about 390 post-staining that  v i s u a l i z e d by  t h i s method.  negative s t a i n seemed to be type of s t a i n .  diameter of 390 p e r c e n t PTA diameter.  pH  8.0  and  Two  nm,  at pH  nm  Fixation  showed an and  UGV  highly  p a r t i c l e was  at pH  not  7.0  w i t h pH  6,0  conditions  revealed a  f i x e d diameter.  VLP  Two  showed p a r t i c l e s of about  7.0  been s t o r e d i n b u f f e r  PTA  i n diameter; TIV  s i m i l a r enlargement from 13 9 to 17 2 nm Other authors  suggesting  dependent on pH  p a r t i c l e s which had  to 5 90 nm  nm,  without  Dimensions of p u r i f i e d VLP's i n  p e r c e n t UA  to pH  then s t a i n e d  to about 550  coat of the  i d e n t i c a l to the 6.0  glutaralde-  edge-to-edge  sectioning  showed a diameter of about 350  an outer l a y e r of the  the  as w e l l ) b e f o r e negative  sectioning  nm.  pH's.  as good i n most cases.  V i r u s - l i k e p a r t i c l e s which were f i x e d w i t h  and  in  Measurements were made under a  shown, i n which u r a n y l a c e t a t e (PTA)  c e l l s and  enlarged even  480 at  further  p a r t i c l e s showed a a f t e r storage a t pH  8.0.  ( B e l l e t t 196 8) have a l s o r e p o r t e d enlargement of  -67TABLE IX —  UGV Measurements  Dimension measured  Fixation  Stain and pH  Storage b u f f e r Number measured and pH  none  0.05 M T r i s HCI, pH 8.0  Mean Length (nm) ± standard deviation  42  355 ± 39  29  389 ± 37  UGV diameter in vitro  glut  UGV diameter in situ  glut/ 0s0  UA/Pb  UGV diameter in vitro*  glut  UA,pH 6 . 0 0.01 M Na a c e t a t e pH 6.0  390  UGV diameter in v i t r o  glut  PTA, pH 7.2  0.01 M Na a c e t a t e pH 6.0  390  UGV diameter in vitro  none  PTA pH 5.5  0.01 M KPO b u f f e r , pH 6.0  TIV diameter  none  a  —  4  3  D  II  UGV diameter' none  PTA, pH 7.2  0  II  9 10  II  475 ± 57 139° ± 15 !  490  0.01 M KPO. pH 7.0 4  UGV diameter TIV diameter  none none  PTA 0.05 M T r i s pH 7 . 2 HCI pH 8.0  50 40  595 ± 66 171 ± 17  UGV diameter TIV diameter Latex beads^  none none none  PTA pH 7.2  17 5 20  550 ± 49 173 ± 20 775 ± 62  0.05 M T r i s HCI pH 8.0  S t a i n s and f i x a t i v e s a b b r e v i a t e d as f o l l o w s : PTA i s 2% phosphotungstic a c i d , used as a negative s t a i n ; UA i s 2% u r a n y l a c e t a t e , a l s o a n e g a t i v e s t a i n ; g l u t i s 5% g l u t a r a l d e h y d e , used as a f i x a t i v e w i t h or without OsO., 2% osmium t e t r o x i d e ; and UA/Pb i s u r a n y l a c e t a t e f o l l o w e d by Reynold's lead c i t r a t e s t a i n , used on t h i n s e c t i o n e d m a t e r i a l . Measurements of June 1975 done by Dr. J.A. Dodds. G  P u b l i s h e d TIV diameter i s 130 nm Tremaine 1968).  (Kalmakoff and  ^Latex beads (Sigma) were of average diameter 790 nm a c c o r d i n g t o manufacturer's d e s c r i p t i o n .  T68-,  TIV  p a r t i c l e s f r o m 130 nm t o 170 nm i n n e g a t i v e l y  preparations,  without p r i o r f i x a t i o n .  stained  Individual sides of  t h e UGV h e x a g o n a l p a r t i c l e a l s o e x h i b i t e d s w e l l i n g :  from  2 20 nm i n a s t o r a g e b u f f e r  a t pH 7.0 t o 27 0 nm i n pH 8.0 b u f f e r .  Lengths o f sides o f h i g h l y  swollen  measure a c c u r a t e l y . increase  Tail  lengths  a s much p r o p o r t i o n a t e l y  p a r t i c l e s were d i f f i c u l t t o and d i a m e t e r s p r o b a b l y do n o t  as t h e VLP heads.  were e x t r e m e l y v a r i a b l e , most l i k e l y p u r i f i c a t i o n , w i t h maximum l e n g t h s t a i l w i d t h i s a p p r o x i m a t e l y 45 nm. all  longer  t a i l s , b u t many t a i l s  suggestive  due t o b r e a k a g e  lengths  during  c l o s e t o one m i c r o n ; t h e The "knob" was a p p a r e n t o n  seemed t o h a v e b r o k e n b e t w e e n  t h e knob and t h e head, l e a v i n g o n l y wider t a i l  Tail  a short  stump.  o f c o n t r a c t i l i t y was n e v e r  A shortened,  observed.  Measurements were c a l i b r a t e d by t h e use o f TIV particles (300  (130 nm i n d i a m e t e r ) ,  nm), a n d l a t e x b e a d s  tobacco mosaic v i r u s p a r t i c l e s  (790 nm d i a m e t e r )  (Figure 2 c ) .  M e a s u r e m e n t o f t h e d i a m e t e r s o f 20 l a t e x b e a d s g a v e an a v e r a g e o f 775 nm, a d i f f e r e n c e o f o n l y  2%,  Tobacco mosaic v i r u s  lengths  w e r e t o o v a r i a b l e , d u e t o b r e a k a g e a n d a g g r e g a t i o n , t o be u s e f u l as  s i z e markers. The  in VLP,  d i a m e t e r o f 390 nm f o r UGV f i x e d by  s i t u was c o n s i d e r e d given  glutaraldehyde  t o be most u s e f u l f o r d e s c r i p t i o n o f t h i s  t h e s w e l l i n g observed under v a r i o u s  conditions of  s t a i n a n d pH. B. E l e c t r o n m i c r o s c o p y and enzyme d i g e s t i o n o f p e l l e t e d VLP's P u r i f i e d V L P ' s f r o m J J . g i g a s w e r e f i x e d a n d embedded for  e l e c t r o n m i c r o s c o p y and t h e n examined i n t h i n s e c t i o n .  A  -69r e p r e s e n t a t i v e group of p a r t i c l e s i s shown i n F i g u r e 8a. infected  c e l l s , 5- and  6-  s i d e d p r o f i l e s of the  c o a t were observed w i t h a v a r y i n g f u l l n e s s d i a m e t e r corresponded t o the  390  nm  repeated c e n t r i f u g a t i o n s course of f i x a t i o n and  the  coat.  The  d i a m e t e r of i n t r a c e l l u l a r No  tailed  t h i s method, perhaps due  needed t o r e - e s t a b l i s h  to  the  a pellet in  the  dehydration.  I n i t i a l l y , pelleted  VLP's were examined t o v e r i f y  the p a r t i c l e s which were b e i n g p u r i f i e d were i d e n t i c a l t o o r i g i n a l p a r t i c l e s observed by Mattox e t a l . (1972). measurements and  in  membrane-like  inside  VLP's, or VLP's f i x e d b e f o r e n e g a t i v e s t a i n i n g . p a r t i c l e s were observed by  As  the  Both s i z e  u l t r a s t r u c t u r a l d e t a i l s suggested t h a t  VLP's p u r i f i e d were the  that  same as those d e s c r i b e d i n the  the earlier  paper. The of the UGV  d i s t r i b u t i o n of the major b i o c h e m i c a l components  p a r t i c l e was  d e t e r m i n e d by  s p e c i f i c enzyme  i n t h i n s e c t i o n s of p u r i f i e d p e l l e t s of VLP's. were f i x e d i n f o r m a l i n g l u t a r a l d e h y d e and  structure  P e l l e t s of VLP's  acrolein,  were embedded i n the h y d r o p h i l i c  g l y c o l methacrylate f i x a t i o n and  a l o n e , f o r m a l i n and  (GMA,  Leduc and  embedding had  Bernard 1967).  s p e c i f i c enzymes, and  studied i n a hydrophilic  t o e n c l o s e the  medium, The  altered  configurations tissue  medium t o a l l o w f o r exposure t o  enzymes, which were p r e p a r e d i n aqueous s o l u t i o n s . g e n t l e r f i x a t i o n s and  or  a d u a l purpose; t o r e t a i n m o l e c u l a r  d u r i n g f i x a t i o n i n o r d e r t o m a i n t a i n the  r e c o g n i z e d by  digestion  the use  of GMA  reduced the  c o n t r a s t of s t r u c t u r e s viewed by e l e c t r o n VLP's p r e p a r e d u s i n g t h e s e methods are  to  be  the  Unfortunately,  resolution  microscopy.  shown i n F i g u r e  and  Purified 8b.  -70-  F i g u r e 8.  Enzyme t r e a t m e n t s o f embedded p u r i f i e d VLP's. a) P e l l e t e d VLP's, f i x e d i n g l u t a r a l d e h y d e and osmium t e t r o x i d e and embedded i n Epon; s t a i n e d w i t h u r a n y l a c e t a t e and l e a d c i t r a t e . b) P e l l e t e d VLP's, f i x e d i n f o r m a l i n / a c r o l e i n and embedded i n g l y c o l m e t h a c r y l a t e . Normal s t a i n i n g . c) As i n ( b ) , i n c u b a t e d 10 minutes w i t h pronase. d) As i n ( b ) , i n c u b a t e d two hours i n pronase. e) As i n ( b ) , i n c u b a t e d w i t h RNAse f o r two h o u r s . f ) As i n ( b ) , i n c u b a t e d w i t h DNAse f o r two h o u r s .  -71Enzyme t r e a t m e n t s - a r e Plates  shown i n F i g u r e s  8c and d show t h e r e s u l t s  2-hour d i g e s t i o n w i t h pronase. staining after  area  results and  i n the center  Clearly,  8f  and t h e c o a t  of digestions using (DNase).  seen a f t e r area;  removed  (  from  t o 12 h o u r s ;  effect  buffer  f o l l o w e d by d i g e s t i o n f o r 2 h o u r s  of  one  lipid-digesting  No  d i g e s t i o n was  to  detect  pelleted for  lipids  of l i p i d  In the f i r s t  and c a r b o h y d r a t e s and Asboe-Hansen of thin  p o r t i o n s o f t h e VLP's were  sections.  deal of background  treated with  prior  and embedding  menthanol these  ether  (2:1) b e f o r e  procedures  The  also  assessed.  of the VLP.  were  used  i n  attempts  ruthenium (Hayat  red  1970,  of  staining and  as a f i x a t i o n  technique,  R e s u l t s were i n c o n c l u s i v e .  I n the second method,  two d i f f e r e n t i n Epon.  or stirred pelleting  most  effects  stained, but i n d i s t i n c t l y  dirt.  p r e p a r a t i o n s were  shaken w i t h  tried  1971), both  All  were  a t 37 C.  e l e c t r o n microscopy  method,  was  for staining  to fixation  using  Digestion  the sections i n  448, were  techniques  and  a great  lipase  was  cleared  f o r DNAase t h e  pre-soaking  o f any component  indirect  the presence .  after  enzyme,  observed  other  VLP's  Kobayashi  observed  by a s l i g h t l y  The  (RNase)  of the VLP's  RNAase t r e a t m e n t .  marked  Two  was  10 m i n u t e s  with  were  a r e s h o w n i n 8e  as i n d i c a t e d  observed  protein;  considerably thinner.  the nucleases  DNAase t r e a t m e n t ,  ranged  contained  contents of the VLP's  looked  and  the dense, h e a v i l y  Some d i g e s t i o n o f t h e c o n t e n t s  no d i g e s t i o n was  periods  o f 10-minute  of the p a r t i c l e  long digestions the central  completely  8 c , d, e, and f .  lipid  a r e shown i n F i g u r e  fixation.  with  VLP  solvents  Preparations of  overnight with and  and  VLP's  chloroform: The  9a, b, and c.  results Ether  of  -7 2-  F i g u r e 9.  Sectioned p e l l e t s of UGV, p r e - t r e a t e d with l i p i d solvents. a) No treatment. Usual f i x a t i o n , embedding, and stain. b) P r e - t r e a t e d with e t h e r . c) P r e - t r e a t e d with chloroform/methanol ( 2 : 1 ) .  treatment  (9b) had no marked e f f e c t .  Chloroform/methanol  treatment  (9c) caused e x t e n s i v e d i s r u p t i o n of normal VLP  s t r u c t u r e s , and aggregations of d i s o r g a n i z e d p a r t i c l e s were always seen, w i t h long e x t e n s i o n s of m a t e r i a l resembling the membranous VLP coat. the presence of l i p i d ,  One o t h e r attempt was made t o determine and i s d e s c r i b e d i n the s e c t i o n on the  e l e c t r o p h o r e s i s of p r o t e i n s . C.  Light scattering  correction  O p t i c a l absorbance o f the U_. gigas VLP's from 240 t o 310 nm, c o r r e c t e d f o r l i g h t s c a t t e r i n g absorbance due t o the s i z e of the p a r t i c l e , was determined by the method of Noordam (1973).  By t h i s method a l l absorbance above 310 nm i s considered  to be due t o l i g h t s c a t t e r .  A l i n e i s e x t r a p o l a t e d back below  310 nm u s i n g the slope of the absorbance above 310 nm t o determine the p o r t i o n of the absorbance i n the lower wavelength r e g i o n which i s due t o l i g h t  scatter.  R e s u l t s o f t h i s c o r r e c t i o n are shown i n F i g u r e 10. L i g h t s c a t t e r i n g p r o p e r t i e s vary from p r e p a r a t i o n t o p r e p a r a t i o n , dependent  on the degree of a g g r e g a t i o n of the p a r t i c l e s ;  absorbance due t o l i g h t s c a t t e r v a r i e d from 74% t o 87% of t o t a l absorbance i n d i f f e r e n t p r e p a r a t i o n s . ^  C o r r e c t e d A„,„  ^  r a t i o s were i n the range of 1.20 t o 1.29, showing over the u n c o r r e c t e d v a l u e s of 1.14 t o 1.21.  /A _ or>  26 0nm  2 80nm  little  increase  When a rough  estimate of VLP c o n c e n t r a t i o n based on absorbance was needed, 2 0% o f t o t a l absorbance was c o n s i d e r e d t o be due t o t r u e absorbance by the p a r t i c l e s .  A c o r r e s p o n d i n g f i g u r e of 54%  was used f o r t r u e absorbance by TIV (Kalmakoff and Tremaine  -74-  N M  Figure  10.  A b s o r b a n c e o f o n e UGV s u s p e n s i o n b e f o r e and after correction for light scattering absorbance.  -751968) . D.  determined  Sedimentation  coefficient  The s e d i m e n t a t i o n  coefficient  i n five  separate  s u m m a r i z e d i n T a b l e X. standard  (s„_ ) o f t h e V L P was 20 ,w  centrifugations.  Results are  T I V was u s u a l l y c e n t r i f u g e d a s a  i n t h e second c e l l  of the analytical  '  ultracentrifuge.  An a v e r a g e v a l u e o f 6350 S was o b t a i n e d f o r UGV; T I V a v e r a g e d 2450 S.  The r e p o r t e d s e d i m e n t a t i o n  c o e f f i c i e n t o f T I V i s 2200 S  ( B e l l e t t 1968); o u r h i g h e r v a l u e may r e f l e c t d i f f e r i n g c o n d i t i o n s of c e n t r i f u g a t i o n . in  The s v a l u e f o r UGV was m e a s u r e d a t pH 5.5  0.01 M a c e t a t e b u f f e r a n d a t pH 8.0 i n 0.05 M T r i s - H C I b u f f e r  without  s u b s t a n t i a l d i f f e r e n c e s being observed.  r u n s , however, t h e s e d i m e n t a t i o n m e a s u r e d a t pH's l o w e r resulted  i n extremely  than  In several other  c o e f f i c i e n t c o u l d n o t be  6.0 due t o p a r t i c l e a g g r e g a t i o n ,  rapid pelleting.  pH 5.5 o r pH 8.0 a r e s i m i l a r  Apparently,  which  f r e e VLP's a t  i n t h e p r o p e r t i e s which  affect  sedimentation, E.  D e n s i t y i n s u c r o s e o f UGV  The c o n c e n t r a t i o n o f c e s i u m  c h l o r i d e necessary f o r  e q u i l i b r i u m g r a d i e n t c e n t r i f u g a t i o n was f o u n d d i s r u p t UGV p a r t i c l e s .  I n t h e o n l y cesium  t o a g g r e g a t e and  chloride equilibrium  r u n w h i c h was d o n e , t h e c a l c u l a t e d d e n s i t y f o r T I V was 1.32 g / m l , i n agreement w i t h r e p o r t s i n t h e l i t e r a t u r e  ( G l i t z e t a l . 196 8).  The d e n s i t i e s o f UGV, T I V , a n d BSMV ( b a r l e y s t r i p e m o s a i c v i r u s ) w e r e a l s o m e a s u r e d i n 45 t o 90% s u c r o s e g r a d i e n t s w h i c h h a d equilibrated cesium  f o r 24 h o u r s ,  chloride.  a less accurate determination thai i n  R e s u l t s a r e shown i n F i g u r e 11.  After  20 h o u r s  -76-  10 Fraction number  F igure  11.  15  20  E q u i l i b r i u m c e n t r i f u g a t i o n o f UGV, T I V , a n d BSMV in 45-90% s u c r o s e g r a d i e n t s , Sucrose concentrations d e t e r m i n e d by r e f r a c t o m e t r y .  25  -77TABLE X —  Sedimentation  c o e f f i c i e n t of UGV S  20.  w  Buffer conditions  rpm  TIV  0.01 M a c e t a t e pH 5. 5  4059  2300  6400  0.01 M a c e t a t e pH 5. 5  4059  2340  6200  0.01 M a c e t a t e pH 5. 5  4059  not done  6000  0.05 M T r i s - H C l pH 8 .0  4059  2700  6650  0.05 M T r i s - H C l pH 8 .0  6166  2450  6450  values:  2450  6340  Average  TABLE XI —  proteih  band  S  20  ,w  UGV  UGV p o l y p e p t i d e s d e t e c t e d by SDS-polyacrylamide electrophoresis  Molecular  weiaht  Observation o f p r o t e i n bands Tube crels Slab a e l Per cent polyaerylamide 5% 7.5% 10% 10%  -  l  26,000 d a l t o n s  +  2  30,000  3  32,000  -  -  4  35,000  +  -  +  5  37,000  -  + +  +  +  6  40,000  +  -  +  7  42,000  -  +  8  45,000  +  9  53,000  10  64,000  +  +  -  -  -  + + +  +  +  +  +  +  +  +  +  +  -  -78of  centrifugation,  at  1.265  g / m l , a n d UGV  determined sucrose CsCl,  TIV occurred  be due t o i o n i c  o r may  centrifuged somewhat  be a r t i f a c t u a l , to equilibrium.  denser  F.  than  UGV  polyacrylamide  TIV  The  lowered  effects  urea,  SDS,  a t room  bands p r e s e n t  cylindrical  gels.  d a l t o n s was  noted  Table  i f these Particles  g r a d i e n t s had n o t o f UGV  particles  i s shown As  material  polypeptides  by  t o be  with  o r by  1 N HCl.  i n a buffer  incubation polypeptide  b y e l e c t r o p h o r e s i s i n t h e s l a b g e l ; many o f  i n this  g e l were a l s o r e s o l v e d i n t h e  A predominant polypeptide i n a l l g e l runs.  scan  i n Figure  negative  45,000  12.  A  of the slab  b i o c h e m i c a l w o r k w i t h UGV,  i n protein analysis:  component w h i c h would  of  13.  f o r a n a l y s i s were m i n i m a l .  f r o m UGV,  species  R e s u l t s are summarized i n  of a photographic  i n a l l other  due t o t h e l i m i t e d  Ten  7.5,  slab g e l .  f o r 90 s e c o n d s  and m e r c a p t o e t h a n o l ,  SDS-  g e l s o f 5,  and on a 10% p o l y a c r y l a m i d e  temperature  i n Figure  available  encountered  appear  were analyzed  X I and a r e p r e s e n t a t i v e g e l i s shown  microdensitometer gel  d e n s i t y of TIV i n  gel electrophoresis i n cylindrical  bands were d e t e c t e d the  (densities  increasing the density i n  The V L P ' s w e r e d i s s o c i a t e d by b o i l i n g  overnight  g/ml  particles.  o f UGV  10% p o l y a c r y l a m i d e  containing  1,30  proteins  Proteins  and  at approximately  by r e f r a c t o m e t r y ) .  may  a s a b a n d a t 1.2 8 g / m l , BSMV  producing  incomplete  the gels,  of starting  problems  disruption  a high molecular  not penetrate  amount  Two  amounts o f  of  weight  and f a i n t  material.  were  bands  Many o f t h e  -79-  F i g u r e 12.  P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of UGV p o l y p e p t i d e s , i n . a 10% p o l y a c r y l a m i d e s l a b gel. S l o t 1: D i s s o c i a t e d VLP's, no p r e t r e a t m e n t . S l o t 2: VLP's p r e t r e a t e d w i t h c h l o r o f o r m / methanol b e f o r e d i s s o c i a t i o n . S l o t 3: C o n c e n t r a t e d c h l o r o f o r m / m e t h a n o l extract.  71a  I  45,000 d  Figure  13.  M i c r o d e n s i t o m e t e r scan o f UGV p o l y p e p t i d e s , s t a i n e d w i t h Coomassie b l u e s t a i n a f t e r e l e c t r o p h o r e s i s in S D S - p o l y a c r y l a m i d e s l a b gel (10%). Scan of s l o t (1) in F i g u r e 12.  -81bands noted  c o u l d not be seen i n g e l photographs or by  n e g a t i v e s , but only by d i r e c t o b s e r v a t i o n of the g e l . measurements of band p o s i t i o n f o r c a l c u l a t i o n s of  scanning All  molecular  weights were made from measurements of the g e l s themselves; photographs were not used f o r t h i s purpose. The  s l a b g e l e l e c t r o p h o r e s i s was  used f o r d i r e c t  comparison of the p o l y p e p t i d e components of VLP and a f t e r v a r i o u s treatments  samples before  with o r g a n i c s o l v e n t s , to t e s t f o r  the presence of l i p i d s or p r o t e i n s a s s o c i a t e d with l i p i d s e t a l . 1977). VLP  was  No p o l y p e p t i d e bands disappeared  intact  t r e a t e d with . ether or with chloroform/methanol p r i o r to  dissociation.  A f a i n t l y s t a i n i n g , d i f f u s e r e g i o n which  e l e c t r o p h o r e s e d with the b u f f e r f r o n t was ed chloroform/methanol s o l v e n t . or g l y c o l i p i d Figure  when the  (TaS  noted  T h i s r e g i o n may  (Tas e t a l . 1977).  i n the  concentrat-  contain  lipid,  R e s u l t s are i n c l u d e d i n  12. G. N u c l e i c a c i d c h a r a c t e r i z a t i o n Two  methods of c h a r a c t e r i z i n g the UGV  nucleic acid  were used on i n t a c t VLP's, i n a d d i t i o n to s p e c i f i c enzyme digestion:  a c r i d i n e orange s t a i n i n g and the  diphenylamine and o r c i n o l t e s t s . g i v e c o n s i s t e n t r e s u l t s f o r UGV of UGV  standard  The b i o c h e m i c a l t e s t s d i d not and TIV;  however, some assays  p a r t i c l e s were weakly p o s i t i v e i n the diphenylamine t e s t  and a l l o r c i n o l t e s t s of UGV  were n e g a t i v e .  kinds of assay are summarized i n Table X I I . s t a i n i n g r e s u l t s suggest nucleic acid.  The  t h a t UGV  contains a  R e s u l t s of the The  two  a c r i d i n e orange  double-stranded  s t a i n does not d i s t i n g u i s h between  double-  TABLE XII -- N u c l e i c a c i d c o l o r i m e t r i c  Virus  standard  Nucleic acid type  Diphenylamine results  UGV  not known  +  TIV  ds DNA  +  CaMV  tests  a  Orcinbl results -  Acridine orange fluorescence color Green Red +  -  +  ds DNA  +.  -  +  -  TMV  ss RNA°  -  +  -  +  BMV  ss RNA  ±  +  -  +  b  Double-stranded  Cauliflower  DNA.  mosaic v i r u s .  Single-stranded  RNA.  -83stranded RNA and double-stranded H.  Nucleic  Nucleic  DNA.  acid p u r i f i c a t i o n  a c i d was s u c c e s s f u l l y p u r i f i e d from UGV  p r e p a r a t i o n s a t 15 t o 30 ug/ml o f u n d i s s o c i a t e d corrected TIV,  f o r light scatter ^  particles,  (A„,_ times 0.2). By analogy with 260nm 1  about 15% of the n o n - l i g h t  t)  s c a t t e r i n g absorbance i s due  to the n u c l e i c a c i d o f the p a r t i c l e , so a n u c l e i c  acid  p u r i f i c a t i o n from the samples above, i f no n u c l e i c a c i d were l o s t and the f i n a l volume of n u c l e i c a c i d was i d e n t i c a l t o the s t a r t i n g volume o f VLP's, would have a c o n c e n t r a t i o n 4.5 ug/ml.  of 2.3 t o  Since t h i s was normally the maximum amount of v i r u s  a v a i l a b l e , the n u c l e i c a c i d o f t e n r e q u i r e d again, c h a r a c t e r i z a t i o n s  concentration,  and  of the n u c l e i c a c i d were done a t the  l i m i t s o f s e n s i t i v i t i e s of the t e s t s and instruments used. Rather v i g o r o u s d i g e s t i o n with pronase, i n the presence of 1% SDS, was necessary f o r d i s r u p t i o n of VLP's and l i b e r a t i o n of the n u c l e i c a c i d . proteinase Relative by  A m i l d e r p r o t e o l y t i c enzyme,  K, was not e f f e c t i v e i n d i g e s t i n g  the VLP coat.  a b i l i t i e s t o degrade the p a r t i c l e s could be judged  l o s s of opalescence.  The s t a b i l i t y of the UGV p a r t i c l e was  a l s o seen i n i t s r e s i s t a n c e  to disruption f o r protein  analysis;  b o i l i n g i n SDS/urea/mercaptoethanol f o r 90 seconds was not completely e f f e c t i v e . O r c i n o l and diphenylamine t e s t s were repeated purified nucleic acid.  using  The n u c l e i c a c i d from UGV gave a c l e a r  p o s i t i v e r e a c t i o n i n the diphenylamine t e s t , and a negative r e a c t i o n with o r c i n o l ,  Controls  were p u r i f i e d n u c l e i c  acids  from TIV and CaMV (DNA's) and TMV and BMV anomalous r e s u l t s were noted:  (RNA s).  No  1  a l l t h e DNA's gave s t r o n g blue  r e a c t i o n s i n the diphenylamine t e s t and no c o l o r change with o r c i n o l ; and RNA s gave a s t r o n g green c o l o r i n the o r c i n o l 1  tests. Another technique was used t o l i b e r a t e DNA f o r the Kleinschmidt  technique:  a pH 13.0 treatment  required 1 to 2  hours f o r UGV d i s r u p t i o n , i n c o n t r a s t t o a 10 minute exposure f o r CaMV. I.  Buoyant d e n s i t y of UGV-DNA  Cesium c h l o r i d e buoyant d e n s i t y determinations  were  made u s i n g two Beckman Model E u l t r a c e n t r i f u g e s , one a t the A g r i c u l t u r e Canada Research S t a t i o n , and the other a t the Department of M i c r o b i o l o g y , U.B.C., Vancouver. i n Table X I I I .  R e s u l t s are presented  In two runs a t the A g r i c u l t u r e Canada Research  s t a t i o n , UGV-DNA was run alone and w i t h added TIV-DNA as a marker.  Using two c a l c u l a t i o n methods (reference DNA vs no  r e f e r e n c e ) , and t a k i n g TIV-DNA as 1.690 g/ml ( B e l l e t t and Inman 1967), a buoyant d e n s i t y o f 1.714 g/ml was estimated  f o r UGV-DNA.  S e v e r a l measurements of buoyant d e n s i t y u s i n g the Model E a t the M i c r o b i o l o g y Department gave a d e n s i t y of 1.719 g/ml, with M. l y s o d e i k t i c u s DNA as the marker  (1.731 g/ml, S z y b a l s k i 1968).  D i f f u s e and f a i n t minor bands were observed was  i n a l l runs; one which  seen r e g u l a r l y had a d e n s i t y i n the r e g i o n of 1.693*0.003 g/ml.  Scans o f r e s o l v e d e q u i l i b r i u m g r a n d i e n t s , before and a f t e r DNAase d i g e s t i o n , are shown i n F i g u r e 14. 14)  a t 1.719 g/ml disappeared  minor bands d i d not.  The major band  (band 1, F i g .  f o l l o w i n g DNAase d i g e s t i o n ; the  In an e q u i l i b r i u m c e n t r i f u g a t i o n u s i n g  -85-  Figure  14.  E q u i l i b r i u m c e n t r i f u g a t i o n o f UGV-DNA: densitometer s c a n s o f p h o t o g r a p h i c n e g a t i v e s . 1. UGV-DNA; 2. DNA f r o m M i c r o c o c c u s l v s o d e i k t i c u s ; 3, DNA f r o m E.. c o l i . a. B u o y a n t d e n s i t y i n c e s i u m c h l o r i d e . b . As i n (a) , a f t e r DNAse d i g e s t i o n . M.. l v s o d e i k t i c u s m a r k e r DNA r e - a d d e d . c. Buoyant d e n s i t y i n cesium s u l f a t e .  -86-  TABLE XIII — Run #  Buoyant d e n s i t y of UGV-DNA  Marker DNA added Gradient  1  none  Model E  Main band density  Minor band density  CsCl  Agr. Can.  1.713 g/ml  1.699 g/ml  CsCl  Agr. Can.  1.714  Agr. Can.  1.436  2  TIV-DNA  3  E.  4  _M, l v s o deikticus dei  CsCl  M i c r o b i o l . 1.719  1.692  5  M. l v s o .  CsCl  M i c r o b i o l . 1.719  1.698  6.  M. l y s o .  CsCl  M i c r o b i o l . no band  1.694  coli  Cs S0 2  4  a  1 .404  DNAase-digested, marker DNA re-added.  TABLE XIV —  Guanine p l u s c y t o s i n e molar f r a c t i o n of UGV-DNA  = 1.714 - ,1-fifin - 55.1% (Agr. Can.) (G + C) = a - 1.660 g/ml 0.098  =  1  ,  7  1  9  - 1.660 = 60.2% (UBC M i c r o b i o l . )  0 . 098  The symbol a r e p r e s e n t s the buoyant d e n s i t y of the DNA i n q u e s t i o n , ( S c h i l d k r a u t .et a l . 1962) .  -87-  cesium s u l f a t e , two bands were observed: a major band a t 1 . 4 3 6 and a minor one a t 1 , 4 0 4 g/ml.  g/ml  A buoyant d e n s i t y of 1 . 7 1 9 g/ml, by the equation of S c h i l d k r a u t e t a l . ( 1 9 6 2 ) , corresponds t o a molar guanine p l u s c y t o s i n e of 6 0 , 2 % (Table X I I I ) .  f r a c t i o n of  Using the  g r a p h i c a l e s t i m a t i o n method o f E r i k s o n & S z y b a l s k i  (1964), a  cesium s u l f a t e d e n s i t y of 1 . 4 3 6 g/ml corresponds t o a (G + C) molar f r a c t i o n of 70% i f the DNA i s not a t a l l g l u c o s y l a t e d ; i t corresponds t o approximately 60% (G + C) i f the DNA i s 10% glucosylated. J.  N u c l e i c a c i d v i s u a l i z a t i o n by K l e i n s c h m i d t technique  N u c l e i c a c i d molecules i s o l a t e d from p u r i f i e d VLP's were examined by e l e c t r o n microscopy u s i n g t h e K l e i n s c h m i d t technique of spreading n u c l e i c a c i d i n t h i n f i l m s of p r o t e i n . Standards i n c l u d e d f o r comparison were CaMV-DNA, a 2 . 3 t o 2 . 5 micron c i r c l e  (Shepherd and Wakeman 1 9 7 1 ) ; and TIV-DNA,  t h e o r e t i c a l l y a 6 5 micron l i n e a r molecule 1967,  K e l l y and Avery 1 7 4 ) .  double-stranded.  ( B e l l e t t and Inman  Both TIV-DNA and CaMV-DNA a r e  CaMV c i r c u l a r molecules and long TIV molecules  were observed; both were o f a width t y p i c a l of double-stranded molecules  (Figure I 5 ) .  UGV-DNA was i d e n t i c a l t o the other DNA's  i n width, and i s presumably was  double-stranded as w e l l .  always seen i n a l a r g e t a n g l e d form, u s u a l l y with  ends v i s i b l e .  TIV-DNA , free  CaMv p r e p a r a t i o n s showed c i r c u l a r forms and  s h o r t e r l i n e a r molecules, w i t h t h e p r o p o r t i o n o f l i n e a r molecules i n c r e a s i n g w i t h the storage time of p u r i f i e d CaMV-DNA or i n t a c t virions,  as r e p o r t e d  by Shepherd e t a l . ( 1 9 6 8 ,  1970).  -8 8The UGV n u c l e i c a c i d m o l e c u l e s observed by t h i s t e c h n i q u e were l o n g and l i n e a r  ( F i g u r e 15 a ) .  Many were  w e l l - e x t e n d e d by s p r e a d i n g and c o u l d be measured u s i n g a map measuring  d e v i c e on p h o t o g r a p h i c enlargements  a t about 40,000  t i m e s m a g n i f i c a t i o n . The e r r o r f a c t o r due t o t h i s  measuring  procedure was e s t i m a t e d by r e p e a t e d measurements o f one m o l e c u l e , and was found t o be a p p r o x i m a t e l y ± 10%. One  hundred and t h r e e m o l e c u l e s o f UGV-DNA were  measured, i n c l u d i n g e i g h t y - s i x p r e p a r e d by SDS/pronase and seventeen by pH 13.0 t r e a t m e n t . of  l e n g t h s i n microns  A histogram of the d i s t r i b u t i o n  i s i n c l u d e d i n F i g u r e 16.  No d e f i n i t e  peak r e p r e s e n t i n g a modal l e n g t h o f UGV-DNA was found. of  Lengths  DNA m o l e c u l e s v a r i e d from 4 microns t o 36 m i c r o n s ,  c o r r e s p o n d i n g t o DNA's o f m o l e c u l a r w e i g h t s 8 x 10^ t o 72 x 10^ daltons  ( u s i n g an a p p r o x i m a t i o n o f 2 x 10^ d a l t o n s m o l e c u l a r  w e i g h t p e r m i c r o n o f DNA l e n g t h , Lang 1970).  Similar  d i s t r i b u t i o n p a t t e r n s were o b t a i n e d from both methods o f DNA p u r i f i c a t i o n , a l t h o u g h fewer s h o r t m o l e c u l e s were observed w i t h the pH t r e a t m e n t , s u g g e s t i n g t h a t t h e d i s t r i b u t i o n was n o t an a r t i f a c t o f p r e p a r a t i o n methods. The  l e n g t h d i s t r i b u t i o n o f UGV-DNA's was p l o t t e d on  p r o b a b i l i t y paper (Harding 1949)  to test for normality.  Using  t h i s paper, normal d i s t r i b u t i o n s w i l l r e s u l t i n a s t r a i g h t when p l o t t e d by frequency c l a s s e s .  line  Measurements o f CaMV-DNA  were i n c l u d e d , t o a r t i f i c i a l l y i n t r o d u c e a second, known d i s t r i b u t i o n i n t o one a r e a o f t h e range o f v a l u e s f o r UGV-DNA, to  t e s t t h a t a d e v i a t i o n from n o r m a l i t y would be d e t e c t e d u s i n g  t h i s g r a p h i n g procedure  (Figure 16).  With t h e e x c e p t i o n o f t h e  -89-  F i g u r e 15. M o l e c u l e s o f DNA v i s u a l i z e d by t h e K l e i n s c h m i d t technique, a) UGV-DNA; a p p r o x i m a t e l y 15 m i c r o n s . b) CaMV-DNA c i r c l e . c) TIV-DNA b e i n g r e l e a s e d from TIV p a r t i c l e s , a f t e r s p r e a d i n g on 4 M u r e a .  -30-  M5  Mo  h5  L E N G T H (MICRONS)  16.  D i s t r i b u t i o n o f l e n g t h s o f m o l e c u l e s measured o f UGV-DNA a n d CaMV-DNA (118 t o t a l ) . Histogram (dotted a r e a ) and c u m u l a t i v e f r e q u e n c i e s p l o t t e d on p r o b a b i l i t y paper ( l i n e ) .  a r e a o f t h e curve i n c l u d i n g CaMV-DNA l e n g t h s ( r o u g h l y a l l v a l u e s below 3.0 m i c r o n s ) , t h e s e t o f v a l u e s o f UGV-DNA l e n g t h s appear t o be n o r m a l l y d i s t r i b u t e d , i . e . they l i e a l o n g a r e a s o n a b l y s t r a i g h t l i n e on t h e p r o b a b i l i t y graph.  There i s no  s u g g e s t i o n o f a d e v i a t i o n from n o r m a l i t y over t h e range o f 6 microns  t o 25 m i c r o n s , which would argue a g a i n s t t h e presence  of a s i n g l e modal l e n t h .  However, t h e sample s i z e may have  been t o o s m a l l ; t h e r e a r e c l u s t e r s o f l e n g t h v a l u e s around 15 u and 22 u.  A l s o , t h r e e m o l e c u l e s were seen which had l e n g t h s o f  approximately  32 u, were w e l l - s p r e a d and easy t o measure.  N u c l e i c a c i d from UGV p r e p a r e d by t h e a l t e r n a t i v e t e c h n i q u e o f i n c u b a t i o n i n 0.3 N NaOH showed a s i m i l a r v a r i a b i l i t y i n l e n g t h s , w i t h a maximum o f about 32 u. would have been h y d r o l y z e d under t h e s e c o n d i t i o n s .  A l l RNA  The s t r a n d s  o f UGV n u c l e i c a c i d c o m p l e t e l y d i s a p p e a r e d when p r e - i n c u b a t e d with pancreatic deoxyribonuclease  f o r 10 minutes b e f o r e  spreading  CaMV and TIV-DNA's were a l s o d i g e s t e d . An attempt  t o show emergence o f more than one s t r a n d  of DNA from p a r t i c l e s o f UGV by s p r e a d i n g o f i n t a c t VLP's on 4 M urea  (Vasquez and K l e i n s c h m i d t 1968)  r e v e a l e d no e x t r u s i o n o f  n u c l e i c a c i d ; TIV t r e a t e d i n t h e same way i m m e d i a t e l y l o s e DNA i n l o n g , c o i l e d l o o p s  ( F i g u r e 15b).  began t o  Both UGV and CaMV  spread on 4 M u r e a appeared t o l o s e c o a t s t r u c t u r e b u t n u c l e i c a c i d was n o t seen i s s u i n g o u t o f these K.  particles.  R a d i o i s o t o p e l a b e l l i n g o f UGV-DNA  Because f u r t h e r c h a r a c t e r i z a t i o n o f UGV-DNA i n s u c r o s e o r C s C l g r a d i e n t s o r by r e s t r i c t i o n enzyme c l e a v a g e and g e l  e l e c t r o p h o r e s i s r e q u i r e d more DNA than was ever a v a i l a b l e , an attempt was made t o produce t r i t i u m - l a b e l l e d n u c l e i c a c i d .  For  f u r t h e r evidence t h a t UGV p a r t i c l e s c o n t a i n DNA r a t h e r than RNA,  t r i t i a t e d u r a c i l as w e l l as t r i t i a t e d thymidine were s u p p l i e d  as p r e c u r s o r s of both n u c l e i c a c i d s , i n separate c u l t u r e tubes. U r a c i l , the base form, was s u p p l i e d a t 20 uCi/ml of c u l t u r e medium.  Thymidine,  medium a t 5 uCi/ml.  the n u c l e o s i d e , was added t o the c u l t u r e The c h o i c e s - o f the form of the p r e c u r s o r  and the c o n c e n t r a t i o n t o be used were made on the b a s i s of p r e v i o u s l a b e l l i n g s t u d i e s i n v o l v i n g other algae Hanawalt 1972, Dr. L i n Kemp, p e r s o n a l  (Swinton and  communication).  In the f i r s t experiment uptake of the l a b e l l e d p r e c u r s o r s was f o l l o w e d over a two week time p e r i o d . ml t e s t tube c u l t u r e s , seeded w i t h f r e s h l y r e l e a s e d were used t o assay uptake of each prectirsor.  Two 10 zoospores,  Total concentration  of e i t h e r u r a c i l or thymidine was 0.1 u mole/ml c u l t u r e medium. Beginning immediately a f t e r the a d d i t i o n of l a b e l , the tubes were sampled  every two days  (2 x 100 u l from each tube) f o r two weeks.  The uptake of the l a b e l was determined by the f o l l o w i n g calculation: Percent l a b e l absorbed =  maximum counts/100  u l x 10  c o n c e n t r a t i o n of l a b e l (uCi/ml) x e f f i c i e n c y x constant  =  cpm/ml (uCi/ml) x (cpm/dpm) x 2,2 x 10^ (dpm/uCi)  R e s u l t s are shown i n F i g u r e 17a. by U. g i g a s was i n e f f i c i e n t :  Uptake of u r a c i l or thymidine  o n l y 0.6% of the t o t a l  uracil  counts per ml and 0.7% of the t o t a l thymidine counts per ml were  taken up by the a l g a l c e l l s .  The amount of l a b e l i n a l g a l  d i d not i n c r e a s e a p p r e c i a b l y a f t e r one week of growth. were h a r v e s t e d by f i l t r a t i o n a f t e r two weeks growth.  cells  The c e l l s The c u l t u r e  f i l t r a t e , when examined by e l e c t r o n microscopy, c o n t a i n e d b a c t e r i a as w e l l as UGV p a r t i c l e s .  A l i q u o t s of 100 u l p l a t e d  onto n u t r i e n t agar showed e x t e n s i v e b a c t e r i a l growth a f t e r 48 hours.  The f i l t r a t e s were s u b j e c t e d t o sucrose d e n s i t y g r a d i e n t  c e n t r i f u g a t i o n , w i t h the a d d i t i o n of u n l a b e l l e d UGV as c a r r i e r , and f r a c t i o n s were assayed by s c i n t i l l a t i o n c o u n t i n g t o determine if  any UGV p a r t i c l e s had been l a b e l l e d under these c o n d i t i o n s .  R e s u l t s were i d e n t i c a l t o those of the second experiment ( i n which b a c t e r i a l contamination was reduced), which are presented i n F i g u r e 17 b. utilized  Apparently the l a b e l l e d p r e c u r s o r s were not  i n VLP n u c l e i c a c i d s y n t h e s i s t o any d e t e c t a b l e e x t e n t . In the second experiment  c e l l s were grown i n 10 uCi/ml  of u r a c i l or thymidine, and were harvested a f t e r one week, with no.  p r i o r sampling, t o r e s t r i c t b a c t e r i a l contamination.  Algal  c e l l s c o l l e c t e d on f i l t e r s were t r e a t e d with 5% TCA and counted; t o t a l uptake of each l a b e l c l o s e l y p a r a l l e l e d the r e s u l t s of experiment  one.  The f i l t r a t e  the p e l l e t re-suspended  from each tube was p e l l e t e d , and  with added u n l a b e l l e d UGV, and the sample  analysed on sucrose g r a d i e n t s . by e l e c t r o n microscopy  G r a d i e n t f r a c t i o n s were examined  f o r the presence of VLP's and were assayed  f o r r a d i o a c t i v i t y by s c i n t i l l a t i o n c o u n t i n g . i n F i g u r e 17 b.  R e s u l t s are shown  Most counts were d e t e c t e d near the top and the  bottom of the g r a d i e n t s ; UGV p a r t i c l e s were l o c a t e d near the g r a d i e n t c e n t e r s , i n a r e g i o n of e s s e n t i a l l y no counts, f o r e i t h e r u r a c i l or thymidine.  T h i s d i s t r i b u t i o n of VLP's and l a b e l  -94-  Uptake by alga 2.-1  i o  «  H  Q. O  T 4  B. Label in UGV  8  Days after inoculation A -3 H - u r a c i l • -^H-thymidine  Fraction number  Figure  17-  A t t e m p t e d l a b e l l i n g o f t h e n u c l e i c a c i d o f UGV by f e e d i n g w i t h H - u r a c i l a n d H-thymidine. 3  was seen a f t e r sucrose Production  g r a d i e n t a n a l y s i s of e i g h t t e s t c u l t u r e s  of l a b e l l e d UGV-DNA was not apparent u s i n g  method of s u p p l y i n g l a b e l l e d p r e c u r s o r s .  this  No d i f f e r e n c e i n  u t i l i z a t i o n of u r a c i l or thymidine were noted.  -96DISCUSSION I.  Introduction  The eucaryotic work on  isolation  a l g a e has  corallina  proceeded  ( G i b b s e_t a l . 1975,  to  ultrastructural  in  the  field  further  with  the  situ  but  no  had  in purifying  as v i r a l  hosts  Often  from  has  the host  Several authors  been  o f a VLP  they  had  the necessary alga.  a l g a was  nm,  longer  was  being  q u a n t i t y of p a r t i c l e s has  been  as  concerned  of a p o l y h e d r a l v i r u s -  host.  tailed  observed  t o t h e VLP's o v e r UGV  particles  nm  in  appeared  to  be  Toth  c o r e , as n o t e d  and  Wilce  1972,  a  i n other Swale and  in  i n other  100  a m e m b r a n e - l i k e o u t e r c o a t and  1972,  to  JJ. g i g a s showed s t r o n g s i m i l a r i t i e s  non-homogeneous c e n t r a l  (Pickett-Heaps  by  particle  species of algae, p a r t i c u l a r l y  icosahedral, with  collected  o n l y , so  no  observed  This thesis  characterization  VLP's from  400  restricted  have s t a t e d t h a t  t o many o f t h e p o l y h e d r a l p a r t i c l e s  The  the  Chara  t h e h o s t was  a eucaryotic algal  Morphology of the The  been seen  the host  first partial  like-particle  diameter.  of  With  r e p o r t s have as y e t a p p e a r e d , p e r h a p s due  in culturing  structure  algae.  with  S k o t n i c k i e t a l . 1976), a l l  observation.  characterization  difficulties as  algae  for culturing.  attempted,  well  these  infecting  slowly i n comparison  f o r e l e c t r o n m i c r o s c o p i c a l examination  t i m e V L P ' s in  available  but  very  of v i r u s e s  r e s e a r c h d e s c r i b i n g the v i r u s  p u b l i s h e d work on  II.  characterization  the v i r u s e s of p r o c a r y o t i c , blue-green  e x c e p t i o n of the  the  and  densely-packed algal  VLP's  Belcher  1973,  C l i t h e r o e and Hoffman and  Evans 1974,  Markey 1974,  Stanker 1976).  The  Moestrup and  Thomsen  1974,  p u r i f i e d VLP's when p e l l e t e d and  s e c t i o n e d were s i m i l a r i n morphology to those seen i n t h i n s e c t i o n s of i n f e c t e d c e l l s .  A r e c u r r e n t problem i n c u l t u r e  i s o l a t i o n of VLP's from f u n g i , protozoans, and has  been the maintenance of axenic  true i n t r a c e l l u l a r agents of  s t u d i e d , and  contaminants or symbionts.  P a r t i c l e s of UGV  not phages of p r o c a r y o t i c  Because of the l a r g e s i z e  d i s t i n c t i v e morphology of the UGV contamination was  section. gigas  and  particle, bacterial  not as severe a problem i n t h i s are much l a r g e r than any  study.  reported  b a c t e r i o p h a g e s , approaching b a c t e r i a i n s i z e , and intracellular  algae  c u l t u r e c o n d i t i o n s , to ensure  t h a t the VLP's i s o l a t e d r e p r e s e n t the host organism being  eucaryotic  and  no  symbionts of _U. gigas were ever observed i n t h i n  A l s o the p u r i f i e d p a r t i c l e s matched those w i t h i n U_.  c e l l s , described  by Mattox e t al.. (1972) and  seen i n our  l a b , i n both s i z e and morphology. P a r t i c l e s of UGV c a p s i d , and  a fibrillar  substance.  Two  the  solid-  aspects of  the  methods could account f o r seemingly empty space  w i t h i n the c a p s i d :  f i x a t i o n and  embedding procedures c o u l d  or d i s t o r t the contents of the VLP, bacteriophage  ft6  as i n the case of  c o n s t i t u e n t s of d i f f e r i n g s t a i n i n g  (Hayat 1970) , i f t h e i r d i s t r i b u t i o n v/as not  w i t h i n the c a p s i d .  shrink  (Gonzalez e_t al_. 1977) ; or v a r y i n g planes of  s e c t i o n i n g might i n c l u d e VLP reactions  i n f u l l n e s s of  i n c a p s i d c o n s t i t u e n t s , i n c l u d i n g dense,  l o o k i n g m a t e r i a l and preparation  seemed to vary  The  uniform  enzyme d i g e s t i o n experiments suggest t h a t  the extremely dense m a t e r i a l i n s i d e the c a p s i d i s composed of  -98protein.  Cores of other complex v i r u s e s  nucleoprotein,  a n u c l e i c a c i d and  double-stranded DNA  v i r u s e s , e.g.  complexed w i t h the DNA,  are known to  contain  p r o t e i n complex; some SV40, have true  histones  l i k e c e l l u l a r chromosomes.  For  UGV,  the p r o t e i n p o r t i o n of a p o s s i b l e n u c l e o p r o t e i n  complex may  more a c c e s s i b l e during  be  surrounding and  enzyme d i g e s t i o n , or may  bound to the DNA,  be  present  so t h a t p r o t e o l y t i c enzyme  d i g e s t i o n would remove the n u c l e i c a c i d as w e l l ; d i g e s t i o n the  by  s p e c i f i c nuclease alone might be harder to d e t e c t v i s u a l l y .  Deoxyribonuclease d i g e s t i o n r e s u l t e d i n only p a r t i a l of the  core area of the VLP's. The  lipid  clearing  p o s s i b i l i t y t h a t the UGV  component has  p a r t i c l e may  not been e l i m i n a t e d .  Results  types of i n d i r e c t experiments which t e s t e d l i p i d s were ambiguous, and s u f f i c i e n t quantity  lipase  the VLP's were not  Digestion  a  from three  f o r the presence of available in  f o r a d i r e c t assay of l i p i d s  chromatographic methods.  include  using  of p e l l e t e d VLP's w i t h a  (with no a c t i v i t y s p e c i f i c f o r phospho- or g l y c o - l i p i d s )  a f t e r embedding f o r e l e c t r o n microscopy d i d not r e s u l t i n a noticeable  change of u l t r a s t r u c t u r e .  d i f f i c u l t i e s using  lipases  Other workers have  (Hayat 1970).  reported  Treatment of VLP's  w i t h chloroform/methanol caused e x t e n s i v e d i s r u p t i o n of t h e i r structure.  Organic s o l v e n t s  are r o u t i n e l y used to t e s t f o r  the presence of l i p i d s ; many animal v i r u s e s  are rendered non-  i n f e c t i o u s f o l l o w i n g the d i s r u p t i o n of the v i r a l envelope such s o l v e n t s . solvent  However, s t r u c t u r a l a l t e r a t i o n s caused  treatments can be  by  by  seen by e l e c t r o n microscopy even f o r  -99some v i r u s e s known to be f r e e of l i p i d Yasunaka 197 7) .  components  (Amako and  C h l o r o f orm/methanol treatment can  d i s r u p t p r o t e i n c o n f i g u r a t i o n s as w e l l as remove  apparently-  lipid.  Enveloped v i r u s e s a l s o l o s e i n f e c t i v i t y a f t e r treatment with e t h e r , but ether  treatment caused no n o t i c e a b l e u l t r a s t r u c t u r a l  changes i n UGV.  C e r t a i n l y UGV  i s not as s u s c e p t i b l e to  s o l v e n t s as the true enveloped v i r u s e s . c o n t a i n some l i p i d :  But UGV  p a r t i c l e s may  chloroform/methanol, a f t e r being used f o r  e x t r a c t i o n of VLP's, produced a d i f f u s e band i n g e l s , i n the r e g i o n where l i p i d s would run. seen i n g e l s of ether  organic  concentrates,  polyacrylamide  T h i s band was  or i n g e l s of  not  untreated  VLP's. K e l l y and Vance lipid  i n two  concerning  (1973) demonstrated the presence of  i r i d e s c e n t v i r u s e s , a f t e r a ten year  e a r l i e r r e p o r t s of l i p i d  workers had  thought t h a t the  controversy  i n these v i r u s e s .  l i p i d detected  was  due  Other  to  contamination of v i r i o n s w i t h host c e l l membranes d u r i n g purification. enough l i p i d  K e l l y and Vance provided  f o r a s i n g l e b i l a y e r i n s i d e the c a p s i d , and  l i p i d s present host c e l l s .  was  that  the  i n v i r u s e x t r a c t s were d i f f e r e n t from those of  the  S t o l t z (1971, 1973)  ICDV's ( i c o s a h e d r a l cytoplasmic an i n t e r n a l l a y e r of l i p i d . encloses  evidence t h a t there  has  proposed a s t r u c t u r e f o r a l l  deoxyriboviruses)  He p o s t u l a t e d  the inner c a p s i d core, and  which  includes  t h a t the l i p i d  layer  t h a t the angular s h e l l of  ICDV's i s composed of p r o t e i n alone.  Using t h i s model, only  s o l v e n t s which d i s t u r b e d the e x t e r n a l p r o t e i n s t r u c t u r e would g a i n access to the i n t e r n a l membrane l a y e r . s i n g l e l a y e r of s t a i n e d m a t e r i a l l e f t  The  extremely t h i n ,  i n t a c t at the  periphery  -100of UGV  p a r t i c l e s a f t e r lengthy p r o t e o l y t i c d i g e s t i o n  could r e p r e s e n t t h i s l i p i d  layer.  l a y e r does not  r i g i d angularity  capsid;  p r o v i d e the  (Figure  In S t o l t z ' s model, the seen i n the  8c,d)  lipid  intact  the p r o t e a s e - d i g e s t e d VLP's i n t h i s study are c i r c u l a r  in outline,  i n contrast  to the hexagonal p r o f i l e of  intact  particles. The  diameter of the UGV  observed among the date the  eucaryotic  particle  algal viruses  l a r g e s t r e p o r t e d a l g a l v i r u s was  (diameter 240  nm)  (400  (Kelly  Robertson 1973).  The  130  nm  and  the octopus ICDV to a maximum of 26 0 nm,  diameters of the  unclear  as the  The  swine f e v e r  data a v a i l a b l e  suggests t h a t  among the v i r u s e s  included  and  of is  a t y p i c a l of most known a v i r u s group such  such d i v e r s i t y of s i z e s must the  little  amount of  significant differences  i n t h i s group  proteins,  a double-stranded DNA  160  d a l t o n s , and  be  serological  ( K e l l y and  exist  Robertson 197 3),  s i m i l a r i t i e s , an i n t e r n a l l i p i d membrane,  cytoplasm of i n f e c t e d  virus,  Wide v a r i a t i o n i n diameter--from  Christmas 1971)  e s p e c i a l l y since  3,  with intermediate  S e v e r a l workers have suggested t h a t  artificial,  x 10^  frog virus  s i g n i f i c a n c e of t h i s v a r i a b i l i t y  ICDV's which i n c l u d e s  Structural  ICDV's range from  nm--has been r e p o r t e d among known s t r a i n s  (Howse and  viruses.  the  insect iridescent viruses,  diameters from other i n s e c t ICDV's, A f r i c a n  lymphocystis v i r u s .  To  lymphocystis v i r u s of f i s h  and  other amphibian v i r u s e s .  ICDV's.  ICDV's  of the  13 0 to 26 0  or the  largest  largest  were c e r t a i n v a r i a n t s  and  i s the  from Oedogonium  (Rickett-Heaps 1972), and  f o r many of the  nm)  multiple  genome of approximately 130  accumulation of v i r i o n s w i t h i n c e l l s have been designated as  the the  to  -101characteristics The  unifying  UGV  t h i s d i v e r s e group  p a r t i c l e can  apparently change dimensions  under v a r i o u s c o n d i t i o n s of s t a i n and from 390  nm  (in. s i t u  to a maximum of 590 storage at pH  pH:  swelling  nm  Only one  other a l g a l v i r u s has  are  viruses  on N - l and  AS-1  diameter and contractile in  Shilo  viruses,  are  are q u i t e t h i n t a i l s of the  LPP  closer  f o r UGV,  Bacteria  and  the UGV  diameters of AS-1  The  (about 20 nm  and  and  (only  have  are  blue-green Tails  of  non-  long as the  wide).  20 nm  The  capsid  non-  r e l a t i v e l y short long).  c a p s i d and  The 1000  relative nm  tail—  c o n t r a c t i l e - t a i l e d blue-green  evidence of c o n t r a c t i l i t y was  diameter i s 4 to 8 times l a r g e r N-l  the  c o n t r a c t i l e forms, found  v i r u s group are  t a i l of UGV—400 nm  However, no  are  only been  c o n t r a c t i l e or  about twice as  i n p r o p o r t i o n s to the  algal viruses.  be  1973).  comparison to c a p s i d diameter  s i z e s of c a p s i d and are  t a i l has  Tailed particles  ICDV's.  of blue-green algae can (Padan and  tails  appearance of  hosts to a number of t a i l e d p a r t i c l e s .  contractile  be  other t a i l e d VLP's or v i r u s e s  been observed i n e u c a r y o t i c c e l l s . completely unknown among the  been r e p o r t e d to  the  i n negative s t a i n ; the No  be  c a p s i d diameter i n l e n g t h .  r e p o r t e d on  seen i n s e c t i o n s of c e l l s .  algae are  viruses  pH were s p e c i f i e d .  Belcher 1973); the  l e s s than one  Belcher have not  e x t r a c e l l u l a r VLP  after  to e n l a r g e comparisons of s i z e would  (Aulacomonas V L P — S w a l e and  Swale and  stain)  I f other seemingly 'related VLP's and  meaningful only where c o n d i t i o n s of s t a i n and  q u i t e broad and  observed  ( i n phosphotungstic a c i d s t a i n and  have s i m i l a r c a p a c i t i e s  tailed  was  a f t e r f i x a t i o n , or i n u r a n y l a c e t a t e  f  8.0).  (Fenner 1976).  cyanophages.  By  than  ultrastructural  seen the  -102appearance, UGV  p a r t i c l e s have a c a p s i d t h a t shares many-  p r o p e r t i e s with ICDV's, and a t a i l ,  of unknown f u n c t i o n ,  and  v a r y i n g i n appearance from the t a i l s of phages. III.  Intracellular  development  Mattox, Stewart, p a r t i c l e s develop  and F l o y d  i n the nucleoplasm  (1972) r e p o r t e d t h a t  UGV  of i n f e c t e d  cells.  U. gigas  They never observed VLP's w i t h i n the c h l o r o p l a s t , and the nucleus was  the f i r s t c y t o l o g i c a l  a l t e r a t i o n associated  w i t h i n c i p i e n t UGV  development.  infected  i n agreement w i t h these r e s u l t s .  c e l l s was  My  examination  the e a r l i e s t stages of i n f e c t i o n observed n u c l e a r membranes.  As UGV  l y s i s of  of  UGVC e l l s at  (Figure 3e) had  no  assembly progressed, more of the  c e l l u l a r membrane systems degenerated,  u n t i l f i n a l l y even the  c h l o r o p l a s t became d i s o r g a n i z e d . Observations t h a t UGV  development begins i n the area  of the nucleus are i n accord w i t h r e p o r t s on the development of other e u c a r y o t i c a l g a l v i r u s e s of t h i s s t r u c t u r a l  type  Swale and Belcher 1973,  Moestrup and  Thomsen 1974, 1976).  Pearson  and N o r r i s s 1974,  C l i t h e r o e and Evans 1974,  (Lee  and Hoffman and  1971,  Stanker  These r e s e a r c h e r s and others working on i n t r a c e l l u l a r  development of the ICDV's agree t h a t some type of n u c l e a r r o l e i s i m p l i c a t e d i n the VLP  r e p l i c a t i o n p r o c e s s , and t h a t n u c l e a r  degeneration occurs as a r e s u l t of a l l these i n f e c t i o n s ,  whether  v i r u s assembly takes p l a c e i n the nucleus or cytoplasm.  Pearson  and N o r r i s (197 4) (60 nm)  suggested  t h a t the diameter  of the n u c l e a r pore  might be the f a c t o r which allows f o r the e s t a b l i s h m e n t  of a v i r o p l a s m i n the nucleus; p a r t i c l e s  l a r g e r than 6 0 nm would  -103be assembled  i n a v i r o p l a s m i n the cytoplasm.  (1976) has observed  However, Pienaar  100-130 nm p a r t i c l e s i n the n u c l e i of  phytoplankton; the theory o f entrance by n u c l e a r pores may be too  simplistic. Two v i r u s e s which a r e assembled  i n cytoplasmic  v i r o p l a s m s have r e c e n t l y been shown t o r e q u i r e the presence of a nucleus:  f r o g v i r u s 3(FV3—Goorha e t a l . ,1978), and  A f r i c a n swine f e v e r v i r u s  ( A F S V — O r t i n and V i n u e l a 1977).  Enucleated or U V - i r r a d i a t e d host c e l l s w i l l not support v i r u s r e p l i c a t i o n of AFSV or FV3; pulse-chase a u t o r a d i o g r a p h i c s t u d i e s of FV3 r e p l i c a t i o n suggest t h a t a low molecular weight v i r a l DNA i s made i n the nucleus e a r l y i n the i n f e c t i o n c y c l e , and then t r a n s p o r t e d t o the cytoplasm.  Since these v i r u s e s c o n t a i n  double-stranded DNA, as does UGV, n u c l e a r involvement may c o n s i s t o f an enzyme requirement  f o r DNA r e p l i c a t i o n , or simply  the breakdown of host DNA f o r a source of d e o x y r i b o n u c l e o t i d e s . I n v e s t i g a t i o n s of i n t r a c e l l u l a r development of s e v e r a l ICDV's (FV3, lymphocystis, TIV) and s e v e r a l v i r u s e s of e u c a r y o t i c algae have i n c l u d e d o b s e r v a t i o n s of nascent angular forms of incomplete c a p s i d s i n c e l l s a t e a r l y stages of infection  (Yule and Lee 1973).  these incomplete hexagonal  U s u a l l y c e l l s c o n t a i n many of  o r pentagonal  forms, so the  p o s s i b i l i t y t h a t the forms are a s e c t i o n i n g a r t i f a c t i s s l i g h t . These were seen i n germlings of U. gigas i n f e c t e d by UGV as well  (Figure 3 e ) . U gigas and other algae seemed t o be most l i k e l y t o  c o n t a i n VLP's d u r i n g the zoospore l i f e cycles  (see Table I ) .  and germling stages of t h e i r  Pickett-Heaps  (1972) suggested  that  -104t r a n s m i s s i o n of these v i r u s e s might occur o n l y d u r i n g the zoospore stage, when the host a l g a i s not e n c l o s e d i n a t h i c k c e l l wall.  The zoospore without i t s w a l l may  a l s o be more  s u s c e p t i b l e to environmental s t r e s s ; s e v e r a l c e l l d i v i s i o n s a development  sequence  and  i n t o a germling must occur i n a s h o r t  p e r i o d of time subsequent  to zoospore r e l e a s e .  Cvlindrocapsa  g e m i n e l l a , another U l o t r i c h a l e a n a l g a which has been shown to c o n t a i n VLP's  (Hoffman  s u s c e p t i b l e to VLP  and Stanker 1976), seems to be most  i n f e c t i o n — o r most l i k e l y to express a  l a t e n t i n f e c t i o n - - d u r i n g the germling stage, e s p e c i a l l y i f s t r e s s ed by heat shock.  A heat shock treatment of U. g i g a s at the  zoospore and germling stages produced the  number of VLP's r e l e a s e d .  a six-fold  The i n c r e a s e may  increase i n be due t o a  g r e a t e r percentage of a l g a l c e l l s becoming i n f e c t e d ;  alternatively  more c e l l s c o u l d have been induced to show a c t i v e i n f e c t i o n the  heat shock.  My r e s u l t s , though p r e l i m i n a r y , i n d i c a t e  the  e f f e c t of heat shock i s augmented i n the dark.  after  that  Lack of  light  d u r i n g a normal p h o t o p e r i o d would i n c r e a s e the s t r e s s on a heatshocked p h o t o s y n t h e t i c organism, by f o r c i n g u t i l i z a t i o n of cellular  reserves.  In f u t u r e experiments, heat shock treatments  could be done i n c o n j u n c t i o n w i t h a time course t o t e s t the t i m i n g of a c t i v e i n f e c t i o n s . settling  C u l t u r e s of zoospores o n l y ,  zoospores, or germlings, c o u l d be assayed a t v a r i o u s  times a f t e r a heat shock to determine at which stage heat has the g r e a t e s t  shock  effect.  Uronema g i g a s VLP's were seen only i n s e c t i o n e d germling c e l l s , or c e l l s of v e r y young f i l a m e n t s , never i n the expanded c e l l s of mature f i l a m e n t s .  I n f e c t e d c e l l s may  be more  -105f r a g i l e than h e a l t h y c e l l s , i . e . they may  have l o s t p o r t i o n s  of the plasmalemma as v/ell as other c e l l u l a r membranes, and  be  destroyed d u r i n g the f i x a t i o n and embedding p r o c e s s e s , particularly  i f attached at both ends t o the sturdy c e l l s of  the h e a l t h y f i l a m e n t .  At t h i s p o i n t there i s no evidence  mature c e l l s of the f i l a m e n t show i n f e c t i o n with UGV.  that  The  p o s s i b i l i t y t h a t mature c e l l s c o n t a i n the i n f o r m a t i o n f o r VLP s y n t h e s i s i n some form - a s i t u a t i o n  analogous  to lysogeny ,  i n b a c t e r i a - w i l l be c o n s i d e r e d s e p a r a t e l y . The evidence from scanning e l e c t r o n microscopy  of  germlings i s a l s o s u g g e s t i v e of i n c r e a s e d s u s c e p t i b i l i t y  at  t h i s stage of the l i f e c y c l e . similar  t o t h a t of the UGV  Dense spheres of a diameter  p a r t i c l e were observed through  c e l l w a l l s of hog; germlings.  By x-ray m i c r o a n a l y s i s the  the spheres  were found to c o n t a i n more phosphorus than the surrounding cytoplasm;  they may  thus r e p r e s e n t UGV  phosphorus due t o DNA all  content.  algal  p a r t i c l e s , with increased  Since p u r i f i e d UGV  particles  lost  s t r u c t u r e when d r i e d on stubs f o r scanning e l e c t r o n  microscopy,  i t i s u n c l e a r whether these spheres would match  r e f e r e n c e UGV  i n appearance.  Scanning of mature f i l a m e n t s was  not done, but the absence of VLP's i n t h i n s e c t i o n s of such f i l a m e n t s would suggest t h a t they would not have been d e t e c t e d by t h i s method.  The  spheres were seen i n approximately  the hundreds of c e l l s which were scanned. all  stages of the l i f e  microscopy,  5% of  JJ_. gigas c e l l s at  c y c l e , examined by t r a n s m i s s i o n e l e c t r o n  i n c l u d e d about  1% o b v i o u s l y i n f e c t e d c e l l s .  However,  many more c e l l s were examined by scanning than by t r a n s m i s s i o n microscopy,  so comparisons  of f r e q u e n c i e s may  be i n a c c u r a t e .  -106-  The dense spheres  seen by s c a n n i n g microscopy  may i n  f a c t be c o m p l e t e l y d i f f e r e n t e n t i t i e s , t h e s o - c a l l e d polyphosphate  g r a n u l e s , r e p o r t e d by H a r o l d  (1966)  t o be found  i n b a c t e r i a l c e l l s , some f u n g i , and i n a few a l g a e t h a t were examined ( C h l o r e l l a , Scenedesmus, and Chlamydomonas). Polyphosphate  i s b e l i e v e d t o a c t as a s t o r a g e form o f phosphate,  a c c u m u l a t i n g under c o n d i t i o n s u n f a v o r a b l e f o r growth. (1966)  Harold  d e s c r i b e s c y t o l o g i c a l i d e n t i f i c a t i o n o f t h e g r a n u l e s by  use o f b a s i c d y e s , a r a t h e r n o n - s p e c i f i c t e s t , o r by t h e use o f lead s t a i n s .  By e l e c t r o n m i c r o s c o p y ,  the granules stained with  l e a d a r e e x t r e m e l y opaque and may v o l a t i l i z e under t h e e l e c t r o n beam, l e a v i n g c h a r a c t e r i s t i c h o l e s .  Lead s t a i n i n g o f  i n f e c t e d JJ. g i g a s c e l l s was r o u t i n e l y used, and d a r k l y s t a i n i n g p y r e n o i d s were seen w i t h i n c h l o r o p l a s t s ( F i g u r e s l a and l b ) , b u t no v o l a t i l i z a t i o n ever o c c u r r e d .  I f polyphosphate  granules  m a i n l y accumulate under f a v o r a b l e growth c o n d i t i o n s , one would not expect t h e i r e x i s t e n c e , i n q u a n t i t y , i n a f r e s h young c u l t u r e o f m a i n l y 2- t o 4 - c e l l e d f i l a m e n t s . IV.  P u r i f i c a t i o n and c h a r a c t e r i z a t i o n C h a r a c t e r i z a t i o n o f t h e UGV p a r t i c l e depended on  adequate amounts o f p u r i f i e d p a r t i c l e s f o r t h e v a r i o u s methods of a n a l y s i s .  V a r i o u s methods o f i n c r e a s i n g y i e l d o f VLP's  from an a l g a l h a r v e s t were a s s e s s e d i n t h e course o f d e v e l o p i n g a p u r i f i c a t i o n p r o c e d u r e , b u t they c o u l d d o u b t l e s s be improved. The r o u t i n e h e a t - s h o c k i n g o f f r e s h l y - s e e d e d f l a s k s i n c r e a s e y i e l d o f VLP's.  should  A d d i t i o n a l s t r e s s , perhaps i n t h e form  of a l e s s r i c h medium, lengthened  heat shock, o r p r o l o n g e d  t r e a t m e n t might i n c r e a s e t h e y i e l d o f VLP's.  An attempt t o  dark  -107i n c r e a s e the number of VLP's r e l e a s e d by U V - l i g h t treatment  of  the c u l t u r e r e s u l t e d i n e x t e n s i v e f u n g a l contamination. S u f f i c i e n t t e s t i n g of c e l l u l a r d i s r u p t i o n methods, to r e l e a s e more VLP's from a l g a l t i s s u e , was  not done; i n p a r t i c u l a r a  g e n t l e r but more e f f i c i e n t method than those t r i e d , such  as  the use of a Braun homogenizer, might r e s u l t i n the i n c r e a s e d l i b e r a t i o n of VLP's.  The  simple p u r i f i c a t i o n used at present  seems adequate f o r i s o l a t i o n of VLP's from the c u l t u r e medium; a c l a r i f i c a t i o n step would probably be needed i f the c e l l s were homogenized. VLP's kept a t pH  8.0  The  i n 0.05  e f f e c t i v e a t a lower pH  f i n a l steps of p u r i f i c a t i o n with M. T r i s - H C l , might be more  (pH 7.0  s w e l l i n g of the p a r t i c l e s .  algal  t o 7.5), i n order t o l i m i t  P a r t i c l e s may  the  be more u n s t a b l e d u r i  c e n t r i f u g a t i o n steps when they are h i g h l y swollen. P r o p e r t i e s of the i n t a c t UGV  p a r t i c l e which were  s u c c e s s f u l l y assessed were l i g h t s c a t t e r i n g , c o e f f i c i e n t , and d e n s i t y i n sucrose. due  extent of absorbance  to l i g h t s c a t t e r i n g , about 80% of t o t a l absorbance at  260 nm,  and the sedimentation c o e f f i c i e n t of approximately  S c o u l d be expected VLP.  The  sedimentation  on the b a s i s of the very l a r g e s i z e of t h i s  The d e n s i t y i n sucrose should be r e - t e s t e d , as the  run was  6300  first  probably not c e n t r i f u g e d t o e q u i l i b r i u m ; however, an  estimate of 1.32  g/ml,  very c l o s e to the d e n s i t y of TIV,  o b t a i n e d , implying a s i m i l a r d i s t r i b u t i o n of p r o t e i n , a c i d , and p o s s i b l y l i p i d p r o p e r t i e s of UGV The  i n the p a r t i c l e .  XVI.  content of UGV  o b v i o u s l y a f f e c t e d by l a c k of m a t e r i a l .  nucleic  A comparison of the  and TIV i s i n c l u d e d i n Table  a n a l y s i s of the l i p i d  was  was  A definitive  most statement  -108about the presence of l i p i d i n UGV of  the evidence  to  those of host o r i g i n .  cannot be made on the b a s i s  o b t a i n e d , much l e s s a comparison of VLP  lipids  S i m i l a r d i f f i c u l t i e s were i n v o l v e d  i n the a n a l y s i s of the p o l y p e p t i d e components of UGV:  although  ten s p e c i e s of p o l y p e p t i d e s were d e t e c t e d , a l l but three were v i s u a l i z e d i n b a r e l y r e s o l v e d bands. c a p s i d , and by analogy 20 p o l y p e p t i d e s  with such v i r u s e s as TIV, which codes f o r  ( K e l l y and Robertson 1973), one would expect  have d e t e c t e d more bands. complexity,  structural  too l i t t l e m a t e r i a l a p p l i e d to the g e l s  to r e s o l v e other bands.  was  Cyanophages of t h i s  to  w i t h t a i l s , c o n t a i n more than t e n p o l y p e p t i d e s .  There very l i k e l y was  difficult  Based on the s i z e of the  In a d d i t i o n , the UGV  to d i s s o c i a t e , and  particle  was  a residual undissociated fraction  always seen a t the tops of the g e l s , i n s e v e r a l d i f f e r e n t  methods of d i s s o c i a t i o n .  With more UGV  vigorous d i s s o c i a t i o n , one would expect i n t e n s i t y of a l l the bands noted  a v a i l a b l e and a more an i n c r e a s e i n  here, and more bands would  probably be r e s o l v e d . The  n u c l e i c a c i d of the UGV  double-stranded  DNA.  The  evidence  based i s summarized i n Table XV. buoyant d e n s i t y of 1.719 to  g/ml  p a r t i c l e s was  on which t h i s c o n c l u s i o n i s The  DNA  appears to have a  i n cesium c h l o r i d e , corresponding  a molar f r a c t i o n of c y t o s i n e p l u s guanine  percent.  Two  d i f f e r e n t estimates  (G + C) of  1.719  g/ml).  The d e n s i t y value of the major DNA  by more r e l i a b l e techniques  (the use of M_.  found  (1.714  g/ml  band from  the c e n t r i f u g e i n the U.B.C. Department of M i c r o b i o l o g y estimated  60  of buoyant d e n s i t y were  on the two Model E a n a l y t i c a l u l t r a c e n t r i f u g e s used and  shown to be  was  -109-  TABLE XV —  C h a r a c t e r i z a t i o n of UGV n u c l e i c a c i d  T e s t or assay  Result  Type of N.A. I n d i c a t e d  Enzyme d i g e s t i o n of s e c t i o n e d VLP's a) RNAse no d i g e s t i o n b) DNAse d i g e s t i o n of cores  DNA  Diphenylamine  positive  DNA  Orcinol  negative  A c r i d i n e orange staining  green fluoresence  DNA o r RNA  Buoyant d e n s i t y i n CsCl in Cs S0  1.719 g/ml 1.436 g/ml  DNA DNA  main band disappears  DNA  2  4  added DNAse Kleinschmidt appearance  wide l i n e a r bands DNA or RNA  Strandedness  double-stranded  double-stranded  -110l y s o d e i k t i c u s DNA as a marker and the r e f e r e n c e method of c a l c u l a t i o n ) , and t h i s v a l u e was c o n s i s t e n t from run t o r u n , so a buoyant  d e n s i t y of 1.719 g/ml was c o n s i d e r e d t o be the  more a c c u r a t e of the two v a l u e s .  The d i s c r e p a n c y between the  two instruments may be due t o the d i f f e r e n t marker DNA's used. A buoyant determined  d e n s i t y value o f 1.436 g/ml i n cesium s u l f a t e was f o r the major band seen, corresponding f a i r l y  well  with a cesium c h l o r i d e d e n s i t y of 1.719 g/ml, and p o s s i b l y i n d i c a t i n g up t o 10% g l u c o s y l a t i o n of the DNA S z y b a l s k i 1964, S z y b a l s k i 1968).  (Erikson and  A d e n s i t y of 1.719 g/ml i n  C s C l or 1.436 i n Cs2SO^ i s i n the range c h a r a c t e r i s t i c of DNA: RNA molecules are c o n s i d e r a b l y denser and would be p e l l e t e d i n t h i s cesium c h l o r i d e d e n s i t y  range.  S e v e r a l minor bands were u s u a l l y a l s o observed.  One  which was p r e s e n t i n every run had a d e n s i t y o f approximately 1.695  g/ml i n cesium c h l o r i d e , i n c o n s i s t e n t w i t h i t s d e n s i t y  of 1.404 g/ml i n cesium s u l f a t e .  The major band i n cesium  c h l o r i d e disappeared a f t e r treatment w i t h DNAse; t h i s minor band d i d not.  Because of i t s anomalous behavior i n d e n s i t y  g r a d i e n t s and i t s r e s i s t a n c e t o DNAse, t h i s band does not appear to r e p r e s e n t DNA.  Anomalous bands of p o l y s a c c h a r i d e s and  glycogen have been r e p o r t e d by other authors  (Mandel e_t a l . 1968);  the DNA p u r i f i c a t i o n used d i d not i n v o l v e any treatment t o r i d the samples of these compounds. The v i s u a l i z a t i o n of the n u c l e i c a c i d from UGV by the K l e i n s c h m i d t technique r e i n f o r c e d e a r l i e r evidence t h a t the p a r t i c l e c o n t a i n s double-stranded DNA.  The width of the  molecule was i d e n t i c a l t o CaMV-DNA and TIV-DNA, the two double-  -Ills t r a n d e d DNA markers used.  No c i r c u l a r UGV-DNA m o l e c u l e s  were  seen, a l t h o u g h c i r c l e s o f CaMV-DNA were e a s i l y found; TIV-DNA o c c u r r e d as l a r g e t a n g l e d masses, b u t was c l e a r l y a l i n e a r m o l e c u l e , w i t h f r e e ends.  Strands o f UGV-DNA p r e p a r e d  Kleinschmidt technique disappeared o f DNAse b u t n o t on RNAse.  by t h e  a f t e r i n c u b a t i o n on drops  Use o f a h i g h pH t r e a t m e n t  (pH 13.0)  f o r r e l e a s e o f UGV-DNA r e s u l t e d i n t h e same d i s t r i b u t i o n o f l e n g t h s as t h a t seen a f t e r DNA r e l e a s e by SDS/pronase A l a r g e RNA m o l e c u l e ,  treatment.  o r a DNA i n c l u d i n g r i b o n u c l e o t i d e r e s i d u e s ,  would be degraded by t h i s p r o c e d u r e .  C a u l i f l o w e r mosaic  virus  DNA, which c o n t a i n s r i b o n u c l e o t i d e s ( H u l l and Shepherd 1977, V o l o v i t c h e t al.. 1977), d i s a p p e a r e d  when r e l e a s e d by u s i n g  h i g h pH. E s t i m a t i o n o f t h e m o l e c u l a r w e i g h t o f t h e UGV-DNA by the K l e i n s c h m i d t t e c h n i q u e l e n g t h s from a p p r o x i m a t e l y corresponding  was n o t p o s s i b l e .  4 m i c r o n s t o 3 2 m i c r o n s was o b t a i n e d ,  t o DNA's o f m o l e c u l a r w e i g h t s  t o 64 x 1 0 d a l t o n s . 6  A range o f  of approximately 8  As shown i n F i g u r e 16, none o f t h e  i n t e r m e d i a t e l e n g t h s o c c u r r e d as an obvious peak i n a h i s t o g r a m , s u g g e s t i n g a modal l e n g t h w i t h e x t e n s i v e v a r i a t i o n . distribution molecule.  The  seen c o u l d r e p r e s e n t random breakage o f a l o n g  Perhaps t h e t h r e e l o n g DNA m o l e c u l e s  32 m i c r o n s ) a r e t h e n a t i v e DNA and a l l o t h e r s fragments produced d u r i n g DNA p u r i f i c a t i o n .  (about represent  Care was taken  through p u r i f i c a t i o n t o l i m i t breakage, however, and l o n g , seemingly  i n t a c t TIV-DNA m o l e c u l e s  u s i n g t h e same method.  were seen a f t e r  purification  An a c c u r a t e l e n g t h measurement o f 2.3  m i c r o n s was o b t a i n e d f o r c i r c u l a r CaMV-DNA m o l e c u l e s , i n  -112agreement w i t h t h e p u b l i s h e d v a l u e The  (Shepherd and TVakeman 1971).  l o n g UGV-DNA m o l e c u l e s c o u l d r e p r e s e n t end-to-end aggregates  of a s h o r t e r , n a t i v e m o l e c u l e o f a p p r o x i m a t e l y but t h i s type o f a g g r e g a t i o n c o n d i t i o n s used (Davis et. a l .  16 m i c r o n s ,  o c c u r s r e l a t i v e l y r a r e l y under t h e 1971) ,  A t h i r d e x p l a n a t i o n f o r t h e d i s t r i b u t i o n o f DNA l e n g t h s i s t h a t t h e v i r u s does i n f a c t c o n t a i n DNA's o f v a r y i n g lengths.  That t h e m u l t i p l e l e n g t h s may n o t be a r t i f a c t u a l i s  i m p l i e d by o b t a i n i n g s i m i l a r l e n g t h d i s t r i b u t i o n s u s i n g two d i f f e r e n t DNA p u r i f i c a t i o n p r o c e d u r e s .  (The pH 13.0 procedure  d i d not i n v o l v e p i p e t t i n g , phenol, ether e x t r a c t i o n , or o v e r n i g h t d i a l y s i s , which were f e a t u r e s o f t h e o t h e r method; the DNA was r e l e a s e d and immediately microscopy).  used f o r e l e c t r o n  No w e l l - c h a r a c t e r i z e d DNA v i r u s has been r e p o r t e d  t o c o n t a i n v a r i o u s l e n g t h s o f DNA, o r m u l t i p l e p i e c e s o f DNA, although  t h e r e o v i r u s group i s c h a r a c t e r i z e d by c o n t a i n i n g  m u l t i p l e pieces of double-stranded  RNA.  The p r e s e n c e o f more  t h a n one D N A m o l e c u l e c o u l d i n d i c a t e e i t h e r a divided',, genome of UGV o r some type o f d e f e c t i v e p a c k a g i n g p r o c e s s , where by v a r i o u s l e n g t h s o f VLP-DNA, and p o s s i b l y p i e c e s o f h o s t DNA, a r e encapsidated.  S e c t i o n e d UGV p a r t i c l e s always showed some c e n t r a l  "empty space", n o t a f e a t u r e o f most i c o s a h e d r a l v i r u s e s , and perhaps due t o a r t i f a c t u a l e f f e c t s o f d e h y d r a t i o n  and f i x a t i o n .  On t h e o t h e r hand, space w i t h i n a p a r t i c l e c o u l d i n d i c a t e incomplete spreading  p a c k i n g w i t h DNA.  The UGV p a r t i c l e d i d n o t r e a c t t o  on 4 M u r e a as d i d t h e TIV p a r t i c l e s which r e l e a s e d  large whorls of n u c l e i c a c i d , suggesting out under p r e s s u r e  t h a t t h e TIV-DNA pushed  as t h e p r o t e i n c o a t was d e n a t u r e d .  The UGV  -113-  p a r t i c l e s may  c o n t a i n l e s s DNA,  tha;n TIV and most o t h e r DNA  and  less tightly-packed  viruses.  DNA,  There i s no e v i d e n c e t h a t ,  f o r i t s t h r e e times l a r g e r d i a m e t e r , t h e r e i s a p r o p o r t i o n a t e l y g r e a t e r amount of DNA  i n UGV  than i n  Another o b s e r v a t i o n heterogeneity  of DNA  population  t h a t may  s u p p o r t the h y p o t h e s i s  c o n t e n t s w i t h i n UGV  anomalous s e d i m e n t a t i o n density gradients  TIV.  p a r t i c l e s was  the  p r o f i l e of the p a r t i c l e s i n s u c r o s e  (Figure 7a).  A h i g h l y heterogeneous  of p a r t i c l e s i s i m p l i e d by the broad band  a f t e r c e n t r i f u g a t i o n , e s p e c i a l l y when compared t o c e n t r i f u g e d under the same c o n d i t i o n s .  the most dense component of the VLP, varying p a r t i c l e densities.  obtained  TIV  A range of p a r t i c l e  d e n s i t i e s i s suggested by t h i s p r o f i l e .  UGV  of  Varying  the DNA,  amounts of  could lead to  H e t e r o g e n e i t y of DNA  contents i n  p a r t i c l e s c o u l d be t e s t e d by c o l l e c t i n g p a r t i c l e s from the  uppermost ( l e a s t dense) and  lowermost (most dense) r e g i o n s  a s u c r o s e d e n s i t y g r a d i e n t , and  a n a l y s i n g the DNA  of each f r a c t i o n by the K l e i n s c h m i d t The  of  components  technique.,  attempts t o produce l a b e l l e d VLP-DNA by  addition  of t r i t i a t e d t h y m i d i n e or u r a c i l t o the c u l t u r e medium were unsuccessful: and  very l i t t l e  l a b e l was  e s s e n t i a l l y no l a b e l was  absorbed by the h o s t a l g a ,  incorporated  i n t o VLP-DNA.  r e s u l t i s i n agreement w i t h r e s u l t s of o t h e r  labelling  e x p e r i m e n t s f o r a l g a l n u c l e i c a c i d ( S t e f f e n s e n and Chapman e t a l . 1966,  only.  a l g a l c y t o p l a s m may  Sheridan  1965,  Swinton and Hanawalt 1972), a l l of which  r e p o r t t h a t t h y m i d i n e was c h l o r o p l a s t DNA  This  found t o be i n c o r p o r a t e d  into  S e v e r a l a u t h o r s have s p e c u l a t e d  that  the  l a c k t h y m i d i n e k i n a s e , the n e c e s s a r y enzyme  -114f o r i n c o p o r a t i o n o f t h y m i d i n e i n t o DNA, and t h a t i t i s encoded o n l y i n t h e c h l o r o p l a s t genes (Swinton and Hanawalt 1972, Steffensen  and S h e r i d a n 1965) . L a b e l l e d DNA c o u l d be more c o m p l e t e l y  characterized  i n t h e s m a l l amounts a v a i l a b l e from UGV than has been p o s s i b l e w i t h u n l a b e l l e d DNA. One approach t o produce l a b e l l e d UGV-DNA 32 would be t o use P - l a b e l l e d phosphate s a l t s i n t h e c u l t u r e medium. However, t h e l a b e l l i n g p e r i o d r e q u i r e d by t h i s h o s t 32 organism i s r e l a t i v e l y l o n g , and i n c o r p o r a t e d  P c o u l d cause  a u t o l y s i s , o r a r t i f a c t u a l b r e a k s , i n t h e VLP-DNA by t h e time the VLP's were p u r i f i e d .  P u r i f i e d r a d i o a c t i v e DNA c o u l d be  s t u d i e d by two methods: d e n s i t y g r a d i e n t a n a l y s i s f o r m u l t i p l e s i z e s , and d i g e s t i o n u s i n g r e s t r i c t i o n endonucleases f o l l o w e d by polyacrylamide  g e l e l e c t r o p h o r e s i s , t o estimate  the t o t a l  m o l e c u l a r w e i g h t o f UGV-DNA. C l e a r l y , a r e l i a b l e estimate  o f the molecular weight  of UGV-DNA i s e s s e n t i a l f o r c l a s s i f i c a t i o n o f t h i s VLP. The ICDV's a r e q u i t e homogeneous i n t h e i r c o n t e n t o f DNA, w i t h DNA m o l e c u l a r w e i g h t s o f 130 t o 160 x 10^ d a l t o n s .  Blue-green a l g a l  v i r u s DNA's a r e o f m o l e c u l a r w e i g h t s between 27 and 62 x 10 daltons.  M u l t i p l e p i e c e s o f DNA would e s t a b l i s h UGV as t h e f i r s t  member o f a new v i r u s group; a u n i t l e n g t h o f 3 2 m i c r o n s (or 6 4 megadaltons) would suggest s i m i l a r i t i e s w i t h cyanophages; a l a r g e r DNA might imply a r e l a t i o n s h i p w i t h t h e ICDV's. I f f u r t h e r work v e r i f i e s t h e p r e s e n t e v i d e n c e t h a t UGV p a r t i c l e s c o n t a i n v a r y i n g l e n g t h s o f DNA, i n v e s t i g a t i o n o f the o r i g i n o f t h e s e DNA fragments would be o f i n t e r e s t . I n p a r t i c u l a r , h y b r i d i z a t i o n o f UGV-DNA t o JJ.. g i g a s  (host) DNA  -115should be done t o t e s t f o r d i r e c t packaging without  true VLP-DNA s y n t h e s i s .  of host DNA  A d e f e c t i v e phage o f B a c i l l u s  s u b t i l i s , PBSX, l y s o g e n i z e s i t s host c e l l s .  Upon i n d u c t i o n  with mitomycin C the host c e l l s l y s e and phage p a r t i c l e s are produced, even when DNA s y n t h e s i s i s completely (Okamoto e t a l . 1968b). d e n s i t y determinations  inhibited  By DNA/DNA h y b r i d i z a t i o n , buoyant i n cesium c h l o r i d e , and t r a n s f o r m a t i o n  s t u d i e s on the bacterium  using  'phage DNA, the DNA i n the phage  p a r t i c l e s was determined t o be b a c t e r i a l DNA, c u t t o a uniform l e n g t h and packaged i n phage coat p r o t e i n (Okamoto et, a l , 1968a) . T h i s phage i s c o n s i d e r e d t o be d e f e c t i v e i n the s y n t h e s i s of i t s own DNA.  I f UGV were s i m i l a r l y d e f e c t i v e , and l e s s s p e c i f i c i n  i t s c u t t i n g o f host DNA, a range of DNA lengths packaged UGV p a r t i c l e s might r e s u l t .  into  PBSX i s not i n f e c t i o u s s i n c e i t  c o n t a i n s only host DNA; the analogous case f o r UGV might e x p l a i n difficulties  i n demonstrating  t r a n s m i s s i o n and i n o b t a i n i n g  "healthy" c u l t u r e s . The molar f r a c t i o n of G + C does not a i d i n c l a s s i f i c a t i o n of these v i r u s e s .  The ICDV's are very  heterogeneous i n t h i s p r o p e r t y , ranging from 30 to' 60% (G +C); the cyanophages a l s o show g r e a t v a r i a t i o n , from 37 t o 67%. T h i s parameter cannot be used as a measure o f r e l a t e d n e s s o f these v i r u s e s , as has been done f o r higher organisms.  The DNA  of UGV, with a (G + C) f r a c t i o n of 60%, i s not o u t s i d e the range of e i t h e r of the other two v i r u s groups. On the evidence  a v a i l a b l e now, summarized i n Table XVI,  the UGV p a r t i c l e appears t o be d i s t i n c t i v e , b e a r i n g no strong s t r u c t u r a l or b i o c h e m i c a l r e l a t i o n s h i p t o the ICDV's or the  -116-  TABLE XVI —  Comparison of the p r o p e r t i e s of ICDV's, cyanophages, and UGV  Property  TIV  FV3  Size  130  130  (nm)  Other ICDV  a  130260  Tail  Cyanophage  UGV  55-90  390  +  (nonswollen) +  Envelope S  2300  20,w  CsCl density (g/ml)  4450 (MIV)  550, approx.  1.32  1.32  1.48  Polypeptides: number  20  17  10  M.W. of major species  65,000  49,000  6 35 0 1.32  Ether s e n s i t i v e  Lipid component DNA: mol. wt. (x 106 daltons) (G + C) % nature  +  130160  30%  56?  c  45 ,000  +  130160  linear ds  38,000 & 14 ,000  10 detected  linear ds  27-62  3060%  37-67%  linear ds  l i n e a r ds  Data from K e l l y and Robertson 1973. Data from Padan and S h i l o 1973. 'Double-stranded.  60% l i n e a r ds  -117cyanophages.  On  the b a s i s of morphology alone, the c a p s i d  appears to be s i m i l a r to those of the ICDV's, only s u b s t a n t i a l l y l a r g e r than most; but i t a l s o has u n c h a r a c t e r i s t i c of the ICDV's and phages.  suggesting  a  tail,  a r e l a t i o n s h i p with  As there i s d i v e r s i t y w i t h i n the ICDV's and among  cyanophage types  ( K e l l y and  Robertson 1973,  the e x i s t i n g c l a s s i f i c a t i o n s may i n f o r m a t i o n becomes a v a i l a b l e . U. gigas may  represent  Padan and  S h i l o 1973),  be s u b j e c t to r e v i s i o n s as On the other hand, the VLP  new from  the f i r s t c h a r a c t e r i z e d member of a  new  group of v i r u s e s . V.  Biological  considerations  In f u r t h e r study of the r e l a t i o n s h i p of t h i s VLP  to  i t s a l g a l host, a primary concern would be e s t a b l i s h i n g the i n f e c t i v i t y of t h i s p a r t i c l e . whether any  At the moment i t i s u n c l e a r  c e l l s of U. gigas can be shown to be h e a l t h y ,  f r e e of VLP's under any  environmental c o n d i t i o n s imposed.  i s no d e f i n i t i v e evidence  a l g a l zoospores  There  to show whether i n f e c t i o n proceeds  from o u t s i d e the c e l l , by a b s o r p t i o n of f r e e - f l o a t i n g to  or  particles  (or p o s s i b l y other c e l l types) or whether  the i n f o r m a t i o n f o r VLP  s y n t h e s i s i s i n some way  carried  by  a l l c e l l s of U_. g i g a s , with v a r y i n g extents of e x p r e s s i o n .  All  i s o l a t e s d e r i v e d by s i n g l e c e l l t r a n s f e r s have been shown to r e l e a s e VLP's.  The  p o s s i b i l i t y t h a t some k i n d of l a t e n c y  phenomenon i s i n v o l v e d cannot be r u l e d out. The  occurrence  of l a t e n t i n f e c t i o n s of VLP's i n other  algae has been suggested p r e v i o u s l y . working on the C y l i n d r o c a p s a VLP, induce VLP  production  Hoffman and  Stanker  (1976)  a l s o found t h a t heat shock could  i n young, apparently  healthy colonies  and  -118repeated  t r a n s f e r s gave r i s e to seemingly h e a l t h y  which could always be induced  t o produce VLP's.  filaments Markey  (197 4)  a l s o p o s t u l a t e d a dormant i n f e c t i o n from a study of the brown a l g a P y l a i e l l a , i n which VLP's are seen only i n s p o r a n g i a l  cells.  Synthesis of VLP's i s synchronous from c e l l to c e l l i n a sporangium, and  thus u n l i k e l y to be caused by e x t e r n a l i n f e c t i o n .  Markey suggests t h a t i n f e c t i o n of c e l l s without  thick w a l l s -  gametes or zoospores—may occur, but i n d u c t i o n of VLP only happens i n the mature p l a n t . was  found i n Ectocarpus,  t h a t the VLP  and C l i t h e r o e and Evans  i n f e c t i o n i s probably  VLP's l i m i t e d to one  S i m i l a r t i m i n g of  production infection  (1974) f e e l  systemic, with p r o d u c t i o n  c e l l type o n l y , the sporangia.  p o i n t s out t h a t most of the double-stranded  RNA  are mainly l a t e n t , t r a n s m i t t e d by cytoplasmic  Lemke (1976)  v i r u s e s of f u n g i  exchange, and have  multi-component genomes packaged i n m u l t i p l e c a p s i d s . eucaryotes  may  have e v d v ed l e s s dramatic  forms of  speculated  lower  animals.,  on the mechanism of l a t e n c y : whether  some phenomenon l i k e lysogeny VLP  The  viral  i n f e c t i o n than are seen i n the d i s e a s e s of p l a n t s and No author has  i n phages i s i n v o l v e d , or i f the  p a r t i c l e s remain i n t a c t i n some form i n the cytoplasm  algal  of  of  the  cells. F u r t h e r c h a r a c t e r i z a t i o n of the i n f e c t i v i t y of  would r e q u i r e h e a l t h y a l g a l c u l t u r e s as t e s t organisms.  UGV Since  no a u t h e n t i c a t e d h e a l t h y JJ_, gigas i s a v a i l a b l e from c u l t u r e c o l l e c t i o n s , e f f o r t s would have to be made to e i t h e r d e r i v e a h e a l t h y i s o l a t e or to demonstrate i n f e c t i v i t y i n another  host.  S e v e r a l methods c o u l d be t r i e d f o r d e r i v a t i o n of a h e a l t h y c u l t u r e , such as s e l e c t i o n f o r the most vigorous  zoospores  -119(perhaps by g r e a t e r m o t i l i t y i n moving toward a l i g h t  source;  U. gigas zoospores are p o s i t i v e l y p h o t o t a c t i c ) ; growth of i s o l a t e d germlings on agar to look f o r a l t e r e d m o r p h o l o g i c a l c h a r a c t e r i s t i c s which might c o r r e l a t e w i t h i n f e c t i o n ; repeated s i n g l e - c e l l t r a n s f e r from r e p e a t e d l y and C,  heat-shocked  cultures;  p o s s i b l y c u r i n g by a n t i b i o t i c treatments, e.g., as i s r o u t i n e l y done w i t h l y s o g e n i c  difficulty  i n assessing  bacteriophages.  i n f e c t i o n i n i s o l a t e s i s the  r e l i a n c e at p r e s e n t on e l e c t r o n m i c r o s c o p i c a l detection  of VLP  production.  c u l t u r e medium may synthesis  and  not be  release  mitomycin  are  One  absolute  methods f o r  Negative s t a i n i n g of a l i q u o t s  r e l i a b l e i f very low involved  l e v e l s of  (or i f r e l e a s e  of  VLP  is defective).  Examination of a l g a l c e l l s i n t h i n s e c t i o n i s probably more inaccurate  as a measure of how  only very few  c e l l s can be  to p u r i f i e d UGV p u r i f i e d UGV  has  many c e l l s are i n f e c t e d ,  scanned.  Preparation  of an  since antiserum  would s i m p l i f y these t e s t s immeasurably; to date not been a v a i l a b l e i n the q u a n t i t y  needed f o r  antiserum p r o d u c t i o n . I f f u t u r e experiments suggest t h a t l a t e n t i n f e c t i o n s of UGV  do occur i n U. g i g a s ,  systematic i n v e s t i g a t i o n of  conditions  which t r i g g e r a c t i v e i n f e c t i o n s would be of  interest.  Those c o n d i t i o n s  the great  which might occur as cues i n  nature f o r a c y c l e of i n f e c t i o n of the host a l g a would have implications and  Mrsa  f o r c o n t r o l of e u c a r y o t i c  a l g a l blooms.  (1975) found t h a t a s t a b l e e q u i l i b r i u m  a l g a Plectonema boryanum and  of the  blue-green  i t s v i r u s , LPP-1, developed and  most host c e l l s were r e s i s t a n t to severe i n f e c t i o n s lysis).  Cowlishaw  A s i m i l a r a d a p t a t i o n of host a l g a and  (leading  v i r u s may  have  that to  -120developed between U. gigas and UGV, e u c a r y o t i c algae and  and p o s s i b l y other  t h e i r VLP's.  Lemke (1976) p o i n t s out t h a t a demonstration of t r u e i n f e c t i v i t y — n e e d e d to confirm UGV  as a t r u e v i r u s — r e s t s on  s a t i s f y i n g Koch's p o s t u l a t e s , which may  not be p o s s i b l e i n  cases of l a t e n t i n f e c t i o n or o b l i g a t e symbiosis.  Given  the  p o s s i b i l i t y of l a t e n c y as a widespread phenomenon among e u c a r y o t i c microorganisms, the VLP's observed may  never be  amenable to d e s i g n a t i o n as v i r u s e s , i n the u s u a l sense of  the  term. I f l a t e n c y does occur concerning  i n U. g i g a s , a q u e s t i o n a r i s e s  the taxonomic s t a t u s of the a l g a : does the presence of  a l a t e n t UGV  i n f e c t i o n a f f e c t the host's  characteristics?  morphological  V i r u s - l i k e p a r t i c l e s i n other  eucaryotic  microorganisms have been shown t o a l t e r host phenotype, the k i l l e r  systems i n Paramecium and yeast  hypovirulence fungus and  and  e.g.,  (Lemke 1976), and  sometimes colony c o l o r i n the chestnut  the t a k e - a l l fungus of wheat ( M o f f i t t and  blight  Lister  Ramanathan (1964) emphasized t h a t the c h a r a c t e r i s t i c s  separating  Uronema s p e c i e s from U l o t h r i x s p e c i e s were r e l a t i v e l y minor v a r i a b l e a c c o r d i n g t o environmental c o n d i t i o n s . regarded  fragmentation  production  of long f i l a m e n t s due  as a f e a t u r e of many Uronemas.  and  (1974)  to c o n t i n u a l zoospore  I f mature c e l l s of  a l g a l f i l a m e n t can become i n f e c t e d w i t h VLP's and r e l e a s i n g VLP's, fragmentation  Mitra  1975).  c o u l d a l s o be due  can  the  lyse,  to t h i s  process.  C e r t a i n l y , s p e c i e s of U l o t h r i x should be t e s t e d at a l l stages t h e i r l i f e c y c l e f o r s u s c e p t i b i l i t y t o UGV  or t o changes i n  c u l t u r e c h a r a c t e r i s t i c s a f t e r exposure to  UGV.  of  -121-  To s u m m a r i z e , a V L P r e l e a s e d a freshwater  eucaryotic  by c e l l s  a l g a , has been p u r i f i e d  o f Uronema  gigas,  and p a r t i a l l y  characterized.  W h i l e i t s h a r e s some s t r u c t u r a l p r o p e r t i e s  the  cytoplasmic  icosahedral  deoxyriboviruses  o f e u c a r y o t e s and  some w i t h t h e c y a n o p h a g e s o f p r o c a r y o t i c a l g a e , the presence o f a t a i l  with  t h e s i z e and  a r e u n i q u e among v i r u s e s o f  eucaryotes.  The VLP a l w a y s o c c u r s i n c e l l s w i t h d i s r u p t e d membrane s y s t e m s , • and  i t was o b s e r v e d i n y o u n g g e r m l i n g s o n l y , n e v e r i n c e l l s o f  the mature f i l a m e n t .  The c y t o p a t h o l o g i c a l e f f e c t s i n c e l l s  c o n t a i n i n g VLP's s u g g e s t t h a t t h e VLP i s p a t h o g e n i c , b u t infectivity JJ_. g i g a s  o f t h e V L P ' s c a n n o t be d e m o n s t r a t e d w i t h o u t a  culture.  healthy  Methods have been worked o u t f o r growth o f t h e  a l g a t o m a x i m i z e VLP r e l e a s e , and a p r o c e d u r e f o r p u r i f i c a t i o n o f UGV was d e v e l o p e d .  This  s y s t e m now shows p o t e n t i a l f o r f u r t h e r  characterization of the p a r t i c l e i t s e l f , of the p a r t i c l e w i t h  i t s host alga.  and o f t h e i n t e r a c t i o n  In spite of the r e l a t i v e  o b s c u r i t y o f t h e h o s t and i t s l i m i t e d d i s t r i b u t i o n ,  further  d e s c r i p t i o n o f t h e UGV p a r t i c l e and i t s r e l a t i o n t o i t s h o s t may p r o v i d e eucaryotic  a model system f o r s t u d y o f o t h e r microorganisms.  VLP's o f  -122-  LITERATURE CITED  Adolph, K.W. and R.H. Haselkorn. 1971. I s o l a t i o n and c h a r a c t e r i z a t i o n of a v i r u s i n f e c t i n g the blue-green a l g a Nostoc muscorum. V i r o l o g y 46: 200-208. Agrawal, H.W. and J.H.Tremaine. 1972. P r o t e i n s of cowpea c h l o r o t i c m o t t l e , broad bean m o t t l e , and brome mosaic viruses. V i r o l o g y 47: 8-20. Andrews, J.H. 1976. The pathology of marine algae. Rev. 51: 211-253.  Biol.  Amako, K. and Yasunaka, K. 1977. Ether-induced m o r p h o l o g i c a l a l t e r a t i o n of Pf-1 f i l a m e n t o u s phage. Nature 267: 862-863. Baker, J.R.J, and L.V. Evans. 1973. The s h i p - f o u l i n g a l g a Ectocarpus . I. U l t r a s t r u c t u r e and c y t o c h e m i s t r y o f p l u r i l o c u l a r r e p r o d u c t i v e stages. Protoplasma 77: 1-13. B e l l e t t , A l l a n J.D. 1968. The i r i d e s c e n t v i r u s group. V i r . Res. 13: 225-247.  Adv. i n  . and R.B. Inman. 19 67. Some p r o p e r t i e s of deoxyr ibonucle i c a c i d p r e p a r a t i o n s from C h i l o , S e r i c e s t h i s , and T i p u l a i r i d e s c e n t v i r u s e s . J . Mol. B i o l . 25: 425-432. B i s a l p u t r a , T., J.Y. Cheng, F.J.R. T a y l o r , and F . J . A n t i a . 1973. Improved f i l t r a t i o n techniques f o r the c o n c e n t r a t i o n and c y t o l o g i c a l p r e s e r v a t i o n of microalgae f o r e l e c t r o n micros scopy. Can J . Bot. 51: 371-379. B r a d l e y , D.E. 1965. S t a i n i n g of bacteriophage n u c l e i c w i t h a c r i d i n e orange. Nature 205: 1230.  acids  . 19 67. U l t r a s t r u c t u r e of bacteriophages and bacteriocins. B a c t e r i o l . Rev. 31: 230-314. Brakke, Myron K. 1967. M i s c e l l a n e o u s problems i n v i r u s p u r i fication, pp. 119-136 i n K. Maramorosch and H. Koprowski, eds., Methods i n V i r o l o g y volume 2. Academic P r e s s , New York. Brown, R.M. J r . 1972. 17: 243-277.  Algal viruses.  Adv. i n V i r . Res.  Brunt, A.A., F . J . Barton, J.H. Tremaine, and R. Stace-Smith. 1975. The composition of c a u l i f l o w e r mosaic v i r u s p r o t e i n . • J . G e n . V i r o l . 27: 101-106.  -123-  Chapman, C . J . , N. A. Nugent and R.W. Schreiber. 1966. a c i d s y n t h e s i s i n the c h l o r o p l a s t s of A c e t a b u l a r i a terannea. P l a n t P h y s i o l . 41: 589-592.  Nucleic medi-  Chapman, R.L. 1972. F u r t h e r u l t r a s t r u c t u r a l s t u d i e s of n u c l e a r i n c l u s i o n s i n Porphyridium. J . Phycol. 8 (Supple.): 14. . 1973. The p r e s e n c e o f v i r u s - l i k e p a r t i c l e s c e n t r o s o m e s i n t h e M.B. A l l e n s t r a i n of Porphyridium purpureum . J . Phycol. 9 (Supple.): 16.  and  and I. Lang. 197 3. V i r u s - l i k e p a r t i c l e s and n u c l e a r i n c l u s i o n s i n t h e r e d a l g a P o r p h y r i d i u m purpureum (Bory) Drew e t R o s s . J . P h y c o l . 9: 117-122. Chervenka, C H . 1973. A m a n u a l o f methods f o r t h e a n a l y t i c a l ultracentrifuge. S p i n c o D i v i s i o n o f Beckman I n s t r u m e n t s , Inc. Palo A l t o , C a l i f . C l i t h e r o e , S.B. and L.V. E v a n s . 1974. Virus-like particles i n t h e brown a l g a E c t o c a r p u s . J . U l t r a s t r u c t . Res. 49: 211-217. C o h e n , A r t h u r L. 1975. I.- D e f i n i t i o n s and a b b r e v i a t i o n s u s e d i n the c r i t i c a l p o i n t d r y i n g procedure. II. Annotated b i b l i o g r a p h y o f c r i t i c a l p o i n t d r y i n g a p p l i c a t i o n s and t e c h n i q u e s f o r s c a n n i n g and t r a n s m i s s i o n e l e c t r o n m i c r o s c o p y . The Bomar Company, P.O. Box 225, Tacoma, W a s h i n g t o n 98401. C o w l i s h a w , J . and M. M r s a . 1975. Coevolution of a v i r u s a l g a system. Appl. M i c r o b i o l . 29: 234-239. D a v i s , R.W., M. Simon, and N. D a v i d s o n . 1971. Electron micros c o p e h e t e r o d u p l e x methods f o r mapping r e g i o n s o f b a s e s e q u e n c e homology i n n u c l e i c a c i d s . i n L. Grossman and K. M o l d a v e , e d s . , M e t h o d s i n E n z y m o l o g y Volume XXI D, C h a p t e r 31. A c a d e m i c P r e s s , New York. Dodds, J.A., J.R. S t e i n , and S. H a b e r . 1975. Characterization of a v i r u s - l i k e p a r t i c l e i n a filamentous green a l g a . Can. P h y t o p a t h . S o c , Proceedings o f the F o r t y - f i r s t Session. 42: 26. E r i k s o n , R.L. and W. S z y b a l s k i . 1964. The Cs-SO^ e q u i l i b r i u m d e n s i t y g r a d i e n t and i t s a p p l i c a t i o n f o r t h e s t u d y o f T - e v e n phage DNA: g l u c o s y l a t i o n and r e p l i c a t i o n . V i r o l o g y 22: 111-124. Fenner, Frank. 1976. C l a s s i f i c a t i o n and n o m e n c l a t u r e o f v i r u s e s . S e c o n d R e p o r t o f t h e I n t e r n a t i o n a l C o m m i t t e e on Taxonomy o f Viruses. S. K a r g e r , B a s e l .  -124-  F r a e n k e l - C o n r a t , H. and R.R. Rueckert. 1967. A n a l y s i s of p r o t e i n c o n s t i t u e n t s of v i r u s e s . pp. 1-77 i n K. Maramorosch and H. Koprowski, eds., Methods i n V i r o l o g y volume 3. Academic Press, New York. F r i t s c h , F.E. 19 56. The s t r u c t u r e and r e p r o d u c t i o n of algae. pp. 198-208. Cambridge U n i v e r s i t y P r e s s , Cambridge, England.  the  Garwes, D.H., B.V. P i k e , S.G. Wyld, D.W. Pocock, and R.N. Gourlay. 1975. C h a r a c t e r i z a t i o n of Mycoplasmatales v i r u s - l a i d l a w i i 3. J . Gen. V i r o l . 29: 11-24. Gibbs, A., S k o t n i c k i , A.H., Gardiner, J.E., Walker, E.S., M. H o l l i n g s . 1975. A tobamovirus of a green a l g a . V i r o l o g y 64: 571-574.  and  G l i t z , D.G., G.J. H i l l s , and C F . R i v e r s . 1968. A comparison of T i p u l a and S e r i c e s t h i s i r i d e s c e n t v i r u s e s . J . Gen. V i r o l . 3: 209-220. Gonzalez, C.F., W.G. Langenberg, J.L. Van E t t e n , and A.K. Vidaver. 1977. U l t r a s t r u c t u r e of bacteriophage 0 6: arrangement of the double-stranded RNA and envelope. J . G e n . V i r o l . 35: 353-359. Goorha, R., G. M u r t i , A. G r a n o f f , and R. T i r e y . 1978. Macrom o l e c u l a r s y n t h e s i s i n c e l l s i n f e c t e d by Frog V i r u s 3. VIII. The nucleus i s a s i t e of Frog V i r u s 3 DNA and RNA synthesis. V i r o l o g y 84: 32-50. Gromov, B.V. and Mamkaeva, K.A. 1972. E l e c t r o n microscope examination of B d e l l o v i b r i o c h l o r e l l a v o r u s p a r a s i t i s m on c e l l s of the green a l g a C h l o r e l l a v u l g a r e . C y t o l o g y 14: 256-260. G r o s s - B e l l a r d , M., P. Oudet, and P. Schambon. 1973. Isolation of high molecular weight n u c l e i c a c i d from mammalian c e l l s . Eur. J . Biochem. 36: 32-38. Hansen, J.M. 1974. I s o l a t i o n of higher molecular weight DNA from B a c i l l u s cereus T u s i n g p r o t e i n a s e K. Prep. Biochem. 4: 473-488. Hansmaan, Eugene. 1973. Pigment a n a l y s i s . pp. 359-368 i n J . S t e i n , ed. P h y c o l o g i c a l Methods. Cambridge U n i v e r s i t y Press, Cambridge, England. Harding, J.P. 1949. The use of p r o b a b i l i t y paper f o r the graphi c a l a n a l y s i s of polymodal frequency d i s t r i b u t i o n s . J . Marine B i o l . Assoc. 28: 141-153.  -125-  H a r o l d , F.M. 1966. Inorganic polyphosphates i n b i o l o g y : s t r u c t u r e , m e t a b o l i s m , and f u n c t i o n . B a c t e r i d . Rev. 30: 772-794. Haschemeyer, R.M. and R . J . M e y e r s . 1972. Negative staining. pp. 117-143 i n M.A. H a y a t , e d . P r i n c i p l e s and t e c h n i q u e s of e l e c t r o n m i c r o s c o p y . V a n N o s t r a n d R e i n h o l d Company, New Y o r k . H a y a t , M.A. 1970. P r i n c i p l e s and t e c h n i q u e s o f e l e c t r o n microscopy. Volume I . V a n N o s t r a n d R e i n h o l d Company, New Y o r k . H o f f m a n , L.R. and L.H. S t a n k e r . green alga Cylindrocapsa. .  1975. V i r u s - l i k e p a r t i c l e s i n J . P h y c o l . 11 ( S u p p l e . ) : 7. 1976.  the green a l g a Cylindrocapsa. Horne  F  R. W.  1974.  Virus-like  Can.J.Bot.  Virus structure.  particles in  54: 2827-2840.  A c a d e m i c P r e s s , New  York.  Howse, H.D. and J . Y . C h r i s t m a s . 1971. O b s e r v a t i o n s on t h e u l t r a s t r u c t u r e of lymphocystis v i r u s i n the A t l a n t i c croaker, Micropogon undulatus (Linnaeus). V i r o l o g y 44: 211-214. H u l l , R., R . J . S h e p h e r d , a n d J.D. H a r v e y . 1976. Cauliflower m o s a i c v i r u s : an i m p r o v e d p u r i f i c a t i o n p r o c e d u r e and some p r o p e r t i e s of the v i r u s p a r t i c l e s . J . Gen. V i r o l . 31: 93-100. and R . J . S h e p h e r d . 1977. The s t r u c t u r e o f c a u l i f l o w e r m o s a i c v i r u s genome. V i r o l o g y 79: 216-230. K a l m a k o f f , J . and J.H. T r e m a i n e . 1968. perties of Tipula iridescent virus.  Physicochemical proJ . V i r o l . 2: 738-744.  Kazama? F.Y. and K.L. S c h o r n s t e i n . 1972. Herpes-type a s s o c i a t e d with a fungus. S c i e n c e 177: 696-697. K e l l y , D.C. acid.  and R . J . A v e r y . 1974. Frog J . Gen. V i r o l . 24: 339-348.  particles  virus 3 deoxyribonucleic  . 1974. The DNA c o n t e n t o f f o u r s m a l l i r i d e s c e n t v i r u s e s : genome s i z e , r e d u n d a n c y , and homology d e t e r m i n e d by r e n a t u r a t i o n k i n e t i c s . V i r o l o g y 57: 425-435. K e l l y , D.C. and J . S . R o b e r t s o n . 1973. Icosahedral cytoplasmic deoxyriboviruses. J . Gen. V i r o l . 20 ( S u p p l e . ) : 17-41. K e l l y , D.C. and D.E. V a n c e . 197 3. The l i p i d c o n t e n t o f two i r i d e s c e n t v i r u s e s . J . Gen. V i r o l . 21: 417-423.  -126K l e i n s c h m i d t , A.K. 1968. Monolayer techniques i n e l e c t r o n microscopy of n u c l e i c a c i d molecules. pp. 361-376 i n L. Grossman and K. Moldave, eds. Methods i n Enzymology volume XII B. Academic P r e s s , New York. Kobayasi, T. and G. Asboe-Hansen. 1971. Ruthenium r e d s t a i n i n g of u l t r a t h i n s e c t i o n s of human m a s t - c e l l g r a n u l e s . J . Microscopy 93: 55- 60. Lang, D. 1970. M o l e c u l a r weights of c o l i p h a g e s and c o l i p h a g e DNA. I I I . Contour l e n g t h s and molecular weight of DNA from bacteriophages T4, T5, and T7, and from bovine p a p i l l o m a virus. J . Mol. B i o l . 54: 557-565. Leduc, E.H. and W. Bernard. 1967. Recent m o d i f i c a t i o n of the g l y c o l m e t h a c r y l a t e embedding procedure. J . U l t r a s t r u c t . Res. 19: 196-199. Lee, R.E. 1971. Systemic v i r a l m a t e r i a l i n the c e l l s of the freshwater red a l g a S i r o d o t i a t e n u i s s i m a (Holden) Skuja. J . C e l l S c i . 8: 623-631. Lemke, Paul A. 1976. V i r u s e s of e u c a r y o t i c Ann. Rev. M i c r o b i o l . 30: 105-146.  microorganisms.  Loening, V.E. 19 69. The d e t e r m i n a t i o n of the m o l e c u l a r weight of r i b o n u c l e i c a c i d by p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s . Biochem. J o u r . 113: 131-138. L u f t , J.H. 19 61. Improvements i n epoxy r e s i n embedding methods. J . Biophys. Biochem. C y t o l . 9: 409. M a i z e l , J.V. J r . 1971. P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s of v i r a l p r o t e i n s , pp. 179-247 i n H. Koprowski and K. Maramorosch, eds., Methods i n V i r o l o g y volume 5. Academic P r e s s , New York. Mamkaeva, K.A. 1966. S t u d i e s of l y s i s i n c u l t u r e s of C h l o r e l l a . M i k r o b i o l o g i y a 35: 856-859. Mandel, M. and C.L. S c h i l d k r a u t and J . Marmur. 1968. Use of C s C l d e n s i t y g r a d i e n t f o r determining the guanine p l u s c y t o s i n e content of DNA. i n L. Grossman and K. Moldave, eds., Methods i n Enzymology volume XII B. Academic P r e s s , New York. Manier, J.F., Vago, C , D e v a u c h e l l e , G., and J . L. D u t h o i t . 1971. I n f e c t i o n v i r a l e chez l e s Trichomycetes. Comptes Rendus, Acad. S c i . (Paris) S e r i e s D 273: 1241-1243. Manton, I. and B.S.C. Leadbeater. 1974. F i n e - s t r u c t u r a l obs e r v a t i o n s on s i x s p e c i e s of Chrysochromulina from w i l d Danish marine nanoplankton as a whole. Dan. Vidensk. S e l . B i o l . Skr. 20: 1-26.  -127-  Markey, Donald R. 1974. A p o s s i b l e v i r u s i n f e c t i o n i n the brown alga P y l a i e l l a l i t t o r a l i s . Protoplasma 80: 223-232. Markham, R. 1967. The u l t r a c e n t r i f u g e . pp. 1-39 i n K. Maramorosch and H. Koprowski, eds., Methods i n V i r o l o g y volume 2. Academic Press, New York. Mattern, C.F.T., L.S. Diamond, and W.A. D a n i e l . 1972. Viruses of Entamoeba h i s t o l y t i c a . I I . Morphogenesis of the p o l y h e d r a l p a r t i c l e s and the filamentous agent. J . of V i r o l . 9: 342-358. , J . F . Hruska, and L.S. Diamond. 1974. Viruses of Entamoeba h i s t o l y t i c a . V. U l t r a s t r u c t u r e of the p o l y h e d r a l virus V . J . of V i r o l . 13: 247-248. 3 Q 1  Mattox, K.R., Stewart, F., and F l o y d , J . 1972. Probable v i r u s i n f e c t i o n s i n four genera o f green algae. Can. J . M i c r o b i o l . 18: 1620-1621. Maurer, H.R. 1971. D i s c e l e c t r o p h o r e s i s and r e l a t e d techniques of polyacrylamide g e l e l e c t r o p h o r e s i s . Walter D. Gruyter, New York. McAuslan, B.R. and R.N. Armentrout. 1974. The b i o c h e m i s t r y of i c o s a h e d r a l cytoplasmic d e o x y r i b o v i r u s e s . Current T o p i c s i n M i c r o b i o l , and Immunol. 68: 77-105. McClements, W.L. and P. Kaesberg. 1977. S i z e and secondary s t r u c t u r e o f potato s p i n d l e tuber v i r o i d . Virology 76: 477-484. M i t r a , A.K.  1945.  New o b s e r v a t i o n s  on Uronema.  Nature 155: 115.  . 1947. On the s t r u c t u r e and r e p r o d u c t i o n t e r r e s t r e n. sp. Ann. of Bot. N.S. 11: 349-361.  of Uronema  Moestrup, D. and H.R. Thomsen. 197 4. An u l t r a s t r u c t u r a l study o f the f l a g e l l a t e Pyramimonas o r i e n t a l i s w i t h p a r t i c u l a r emphasis on G o l g i apparatus.. a c t i v i t y and the f l a g e l l a r apparatus. Protoplasma 81: 247-269. M o f f i t t , E.M. and R.M. L i s t e r . 1975. A p p l i c a t i o n of a s e r o l o g i c a l screening t e s t f o r d e t e c t i n g double-stranded RNA mycoviruses. Phytopathology 65: 851-859. Noordam, D. 197 3. I d e n t i f i c a t i o n of p l a n t v i r u s e s . Methods and experiments. Centre f o r A g r i c u l t u r a l P u b l i s h i n g and Documentation, Wageningen, The Netherlands. O b i j e s k i , J . F . , A.T. Marchenko, D.H.L. Bishop, B.W. Cann, and F.A. Murphy. 1974. Comparative e l e c t r o p h o r e t i c a n a l y s i s of the v i r u s p r o t e i n s of four r h a b d o v i r u s e s . J . Gen. V i r o l . 22: 21-33.  -128-  Okamoto, K., J.A. Mudd, J . Mangan, W.M. Huang, T.V. Subaiah, and J . Marraur. 1968a. P r o p e r t i e s of the d e f e c t i v e phage of Bacillus subtilis. J . Mol. B i o l . 34: 413-428. , and J . Marmur. 1968b. Conversion of B a c i l l u s s u b t i l i s DNA t o phage DNA f o l l o w i n g mitomycin C induction. J . Mol. B i o l . 34: 429-437. O r t i n , J . and E. V i n u e l a . 1977. Requirement of c e l l nucleus f o r A f r i c a n swine f e v e r v i r u s r e p l i c a t i o n i n Vero c e l l s . J . V i r o l . 21: 902-905. Padan, E. and M. S h i l o . 1973. C y a n o p h a g e s — v i r u s e s a t t a c k i n g blue-green a l g a e . B a c t e r i o l . Rev. 37: 343-370. Palade, G. 1952. A study o f f i x a t i o n f o r e l e c t r o n microscopy. J . Exp. Med. 95: 285. Pearson, B.R. and R.E. M o r r i s . 1974. I n t r a n u c l e a r v i r u s - l i k e p a r t i c l e s i n the marine a l g a Platymonas sp. (Chlorophyta, Pr.asinophyceae) . P h y c o l o g i a 13: 5-9. Pederson, T. 1969. Use of diphenylamine as a c o l o r i m e t r i c f o r r i b o n u c l e i c a c i d . A n a l . Biochem. 28: 35-46.  reagent  P i c k e t t - H e a p s , J.D. 1972. A p o s s i b l e v i r u s i n f e c t i o n i n the green a l g a Oedogonium. J . P h y c o l . 8: 44-47. P i e n a a r , R.N. 1976. V i r u s - l i k e p a r t i c l e s i n t h r e e s p e c i e s o f phytoplankton from San Juan I s l a n d , Washington. Phycologia 15: 185-190. Ramanathan, K.R. 1964. U l o t r i c h a l e s . Indian C o u n c i l o f A g r i c u l t u r a l Research, New D e l h i , I n d i a . Reynolds, E.S. 1963. The use of l e a d c i t r a t e a t h i g h pH as an e l e c t r o n opaque s t a i n i n e l e c t r o n microscopy. J. Cell B i o l . 17: 208. Safferman, R.S. 1973. P h y c o v i r u s e s . i n N.G. Carr and B.A. Whitton, eds., The b i o l o g y of the blue-green a l g a e . Blackwell S c i e n t i f i c P u b l i s h i n g , Oxford, England. pp. 214-237. , T.S. Diener, P.R.Desjardins, and M.E. M o r r i s . 1972. I s o l a t i o n and c h a r a c t e r i z a t i o n of AS-1, a phycovirus i n f e c t i n g the blue-green algae A n a c y s t i s n i d u l a n s and Synechococcus cedrorum. V i r o l o g y 47: 105-113. Safferman, R.S. and M.E. M o r r i s . Science 140: 674-680.  1963.  Algal virus:  isolation.  -129-  Safferman, R.S. and M.E. M o r r i s . 1977. Phycovirus B i b l i o g r a p h y . V i r o l o g y S e c t i o n , B i o l o g i c a l Methods Branch, Environmental M o n i t o r i n g and Support Lab, O f f i c e of Research and Development, U.S. Environmental P r o t e c t i o n Agency, C i n n c i n n a t i , Ohio. EPA 600/9-77-008, May 1977. S c h i l d k r a u t , C.L., J . Marmur, and P. Doty. 1962. Determination of the base composition of d e o x y r i b o n u c l e i c a c i d from i t s buoyant d e n s i t y i n C s C l . J . Mol. B i o l . 4: 430-443. Schnepf, E.C., J . Soeder, and E. Hegewald. 1970. Polyhedral v i r u s - l i k e p a r t i c l e s l y s i n g the a q u a t i c phycomycete Aphelidium sp., a p a r a s i t e of the green a l g a Scenedesmus armatus. V i r o l o g y 42: 482-487. Schuster, F.L. and T.H. Dunnebacke. 1971. Formation of bodies a s s o c i a t e d w i t h v i r u s - l i k e p a r t i c l e s i n the amoeboflagellate N a e g l e r i a g r u b e r i . J . U l t r a s t r u c t . Res. 36: 659-668. Sharp, D.G. 1965. Quantitative in v i r u s research. Methods counting. i n G.F. Baker and E l e c t r o n Microscopy, W i l l i a m Maryland.  use of the e l e c t r o n microscope and r e c e n t r e s u l t s of p a r t i c l e E . H . Z e i t l e r , eds., Q u a n t i t a t i v e and W i l k i n s Company, B a l t i m o r e ,  Shatkin, A.J. 1969. C o l o r i m e t r i c r e a c t i o n s f o r DNA, RNA, and p r o t e i n determinations. i n K. Habel and N.P. Salzman, eds., Fundamental Techniques i n V i r o l o g y . pp. 231-237. Academic Press, New York. Shepherd, R.J. 1970. C a u l i f l o w e r mosaic v i r u s . D e s c r i p t i o n s of P l a n t V i r u s e s , No. 24.  C.M.I./A.A.B.  , R.J. Wakeman, and R.R. Romanko. 1968. c a u l i f l o w e r mosaic v i r u s . V i r o l o g y 36: 150-152.  DNA  in  , G.E. Bruening, and R.J. Wakeman. 1970. stranded from c a u l i f l o w e r mosaic v i r u s . V i r o l o g y 339-347.  Double41:  > and R.J. Wakeman. 1971. O b s e r v a t i o n on the s i z e and morphology of c a u l i f l o w e r mosaic v i r u s d e o x y r i b o n u c l e i c acid. Phytopathology 61: 188-193. Sherman, L.A. and R. Haselkorn. 1970. LPP-1 i n f e c t i o n of the blue-green a l g a Plectonema boryanum. I. E l e c t r o n microscopy. J . V i r o l . 6: 820-833. S k o t n i c k i , A., A.J. Gibbs, and N.G. W r i g l e y . 1976. s t u d i e s on Chara c o r a l l i n a v i r u s . V i r o l o g y 75:  Further 457-468.  -130Spendlove, R.S. 1967. M i c r o s c o p i c techniques. pp. 475-520. i n K. Maramorosch and H. Koprowski, eds., Methods i n V i r o l o g y Volume 3. Academic P r e s s , New York. S t e f f ^ n s e n , D.M. and W.F. Sheridan. 1965. I n c o r p o r a t i o n of H-thymidine i n t o c h l o r o p l a s t DNA of marine a l g a e . J . C e l l B i o l . 25: 619-627. S t e i n , Janet R. 1973. Handbook of P h y c o l o g i c a l Methods. Cambridge U n i v e r s i t y P r e s s , Cambridge, England. S t o l t z , D.B. 1971. The s t r u c t u r e of i c o s a h e d r a l c y t o p l a s m i c d e o x y r i b o v i r u s e s . J . U l t r a s t r u c t . Res. 37: 219-239. . 1973. The s t r u c t u r e of i c o s a h e d r a l c y t o p l a s m i c deoxyriboviruses. I I . An a l t e r n a t i v e model. J . U l t r a s t r u c t . Res. 43: 58-74. Sueoka, N. 1961. V a r i a t i o n and h e t e r o g e n e i t y of base composition of d e o x y r i b o n u c l e i c a c i d s : a c o m p i l a t i o n of o l d and new d a t a . J . Mol. B i o l . 3: 31-40. Swale, E.M.F. and J.H. B e l c h e r . 1973. A l i g h t and e l e c t r o n m i c r o s c o p i c study of the c o l o u r l e s s f l a g e l l a t e Aulacomonas Skuja. Arch. M i k r o b i o l . 92: 91-103. Swinton, D.C. and P.C. Hanawalt. 1972. In. v i v o l a b e l l i n g of Chlamydomonas c h l o r o p l a s t DNA. J. Cell Biol. 54: 592-597. S z y b a l s k i , W. 1968. Use of cesium s u l f a t e f o r e q u i l i b r i u m density gradient centrifugation. i n L. Grossman and yK;, Moldave, eds., Methods i n Enzymology Volume XII B, pp. 330-341. Tas, P.W.L., M.L. Boerjan, and D. P e t e r s . 1977. The s t r u c t u r a l p r o t e i n s of tomato s p o t t e d w i l t v i r u s . J . Gen. V i r o l . 36: 267-279. Tikhonenko, M.C. and N.B. Z a v a r z i n a . 1966. l y t i c agent of C h l o r e l l a pyrenoidosa.  Morphology of the M i k r o b i o l o g i y a 850-851.  Toth, R. and R.T. W i l c e . 1972. V i r u s - l i k e p a r t i c l e s i n the marine a l g a Chorda tomentosa, Lyngbye (Phaeophyceae) J . P h y c o l . 8: 126-130. Vasquez, C. and A.K. K l e i n s c h m i d t . 1968. E l e c t r o n microscopy of RNA strands r e l e a s e d from i n d i v i d u a l r e o v i r u s p a r t i c l e s . J . Mol. B i o l . 34: 137-147. V o l o v i t c h , M., J.P.Dumas, G. Drudgeon, and P. Yot. 1977. S i n g l e - s t r a n d e d i n t e r r u p t i o n s i n c a u l i f l o w e r mosaic v i r u s DNA. i n L. Bogorad and J.H. W e i l , eds., A c i d e s n u c l e i q u e s e t synthese des p r o t e i n e s chez l e s vegetaux. Colloques i n t e r n a t i o n a u x du Centre de l a Recherche S c i e n t i f i q u e , No. 261. P a r i s , France.  -131W e i n t r a u b , M. , H.W.J. R a g e t l i , and M. V e t o . 1969. The u s e o f g l y c o l methacrylate f o r the study o f the u l t r a s t r u c t u r e of v i r u s - i n f e c t e d l e a f c e l l s . J . U l t r a s t r u c t . R e s . 26: 197-215. Younghusband, H.B. and R.B. Inman. 1974. The e l e c t r o n o f DNA. Ann. Rev. B i o c h e m . 43: 605-616.  microscopy  Y u l e , B.G. and P.E. L e e . 1973. A c y t o l o g i c a l and i m m u n o l o g i c a l study of T i p u l a i r i d e s c e n t v i r u s - i n f e c t e d G a l l e r i a m e l l o n e l l a l a r v a l h e m o c y t e s . V i r o l o g y 51: 409-423. Z a v a r z i n a , N.B. 1961. A l y t i c agent i n c u l t u r e s of C h l o r e l l a pyrenoidosa, Pringh. D o k l a d y A k a d . Nauk. SSSR 137: 435-437. . 1964. sence of b a c t e r i a .  L y s i s o f C h l o r e l l a c u l t u r e s i n t h e abM i k r o b i o l o g i y a 33: 561-564.  and A . E . P r o t s e n k o . 1958. On l y s i s o f a c u l t u r e of C h l o r e l l a pyrenoidosa, P r i n g h . D o k l a d y A k a d . Nauk. SSSR 122: 935-939.  

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