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Gene mutations affecting nuclear behaviour in Paramecium tetraurelia Morton, Phyllida Mary Barry 1977

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GENE MUTATIONS AFFECTING NUCLEAR BEHAVIOUR IN PARAMECIUM TETRAURELIA  PHYLLIDA MARY BARRY MORTON B.Sc,  U n i v e r s i t y o f V i c t o r i a , 1974  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the department of Zoology  We accept t h i s t h e s i s as conforming required  t o the  standard  THE UNIVERSITY OF BRITISH COLUMBIA September, 1977 P h y l l i d a Mary Barry Morton, 1977  In p r e s e n t i n g  t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the  requirements f o r an advanced degree a t the U n i v e r s i t y of B r i t i s h Columbia, I agree t h a t the l i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference  and study.  I further  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my department or by h i s r e p r e s e n t a t i v e s . that  I t i s understood  copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l  gain s h a l l not be allowed without my w r i t t e n  Department of ^oology The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8, Canada V6T 1W5  Date  ayWlrwr  ^  permission.  ABSTRACT  Three Paramecium t e t r a u r e l i a c e l l l i n e s with abnormal p a t t e r n s of n u c l e a r behaviour at c e l l d i v i s i o n and n u c l e a r r e o r g a n i z a t i o n were recovered following nitrosoguanidine  i n separate  mutagenesis.  Two  at experiments  of these l i n e s  c a r r i e d n o n - a l l e l i c tarn mutations, (tarn A and tarn G).  Tarn  i s a s e r i e s of r e c e s s i v e genes t h a t exert a p l e i o t r o p i c e f f e c t on macronuclear behaviour d u r i n g c e l l d i v i s i o n on the m i g r a t i o n and  attachment of trich'ocysts during  t r i c h o c y s t morphogenesis (Ruiz, et a l . , 1976).  At  binary  f i s s i o n the m a c r o n u c l e i of homozygous tarn c e l l s f a i l elongate  and  and f a i l t o migrate t o the d o r s a l c o r t e x .  to This  l e a d s t o unequal d i v i s i o n of the macronucleus or, i n extreme i  V  cases,  complete absence of d i v i s i o n of the macronucleus.  At c y t o k i n e s i s , i n the l a t t e r event, the e n t i r e p r e f i s s i o n macronucleus i s r e t a i n e d by one proter.  s i s t e r c e l l , u s u a l l y the  At n u c l e a r r e o r g a n i z a t i o n , m i s s e g r e g a t i o n  macronuclear anlagen commonly occurs  of the  e i t h e r at the  or at the second p o s t - r e o r g a n i z a t i o n a l c e l l  first  division.  Exautogamonts or exconjugants t h a t do not r e c e i v e a macron u c l e a r anlage at c e l l d i v i s i o n undergo macronuclear regeneration  (Sonneborn, 1940;  d i s t r i b u t i o n of m i c r o n u c l e i  Berger, 1973a).  Improper  occurs f r e q u e n t l y d u r i n g  d i v i s i o n i n c e l l s e x p r e s s i n g the tarn phenotype. ii  The  cell  t r i c h o c y s t s of homozygous tarn c e l l s are abnormally are unattached  and do not d i s c h a r g e .  recovered i s unable  The t h i r d  shaped,  variant  t o complete c o n j u g a t i o n owing t o abnormal-  i t i e s i n the m i c r o n u c l e a r  events.  TABLE OF CONTENTS  ABSTRACT  i i  LIST OF TABLES  .  LIST OF FIGURES  vi viii  LIST OF PLATES  x  ACKNOWLEDGEMENT  xi  1  INTRODUCTION MATERIALS AND METHODS  20  RESULTS  37  SECTION 1.  37  Mutagenesis  SECTION I I . Genetic a n a l y s i s  of the v a r i a n t s .  .  SECTION I I I . Phenotypic a n a l y s i s  42 48  A. Mutations a l t e r i n g the p a t t e r n of macronuclear d i v i s i o n  4$  1.  E f f e c t s on v e g e t a t i v e c e l l s  48  a. Generation time b. E f f e c t s on shape and s i z e distribution  48  c. E f f e c t s on macronuclei  51  d. E f f e c t s on m i c r o n u c l e i  68  e. E f f e c t on t r i c h o c y s t s  79  2.  E f f e c t s during nuclear reorganization  48  .  82  a. Autogamy  82  b. Conjugation  86 iv  V  B. V a r i a n t s w i t h a l t e r e d  micronuclear  distribution  90  1 . E f f e c t s on v e g e t a t i v e c e l l s  90  a. Generation time  90  b. E f f e c t s on the n u c l e i  90  c. Penetrance  92  and e x p r e s s i o n  2. E f f e c t s during nuclear r e o r g a n i z a t i o n .  94  a. Autogamy  94  b. Conjugation  101  DISCUSSION  .,  10,5  CONCLUDING STATEMENT  130  BIBLIOGRAPHY  131  APPENDICES  «•  I . The mode o f a c t i o n I I . Spot  (line  of nitrosoguanidine. .  40-4b ) 2  I I I . The o r i g i n a l s e l e c t i o n system IV. A m i t o s i s PLATES  142 149  152 159 161  LIST OF TABLES  Table. I.  II. III.  Page Mutagenesis experiments: A comparison of the frequency of exautogamous death w i t h the y i e l d ,  ,  41  V a r i a n t l i n e s recovered Exautogamous s e g r e g a t i o n of the tarn genes  IV. V. VI.  VII.  VIII.  IX.  X. XI.  XII.  XIII. XIV.  40  • .  R e s u l t s of mating i n the v a r i a n t 21-3c  ,  .  43 44  Complementation t e s t between tarn genes . .  46  Frequency of complete macronuclear m i s s e g r e g a t i o n i n the tarn A/tam A;am/am double mutant A comparison of the frequency of p a r t i a l and complete macronuclear m i s s e g r e g a t i o n i n tarn A and tarn G  47 61  The e f f e c t of temperature on n u c l e a r m i s s e g r e g a t i o n i n homozygous tarn A and tarn G c e l l s . . . . . . .  67  Comparison of the number of m i c r o n u c l e i between homozygous tarn A and tarn G and wild-type c e l l s  69  Macronuclear  72  s e g r e g a t i o n at c e l l d i v i s i o n .  The frequency of m i s s e g r e g a t i o n of anlagen at the f i r s t and second exautogamous cell division .  87  V a r i a t i o n i n the number of m i c r o n u c l e i i n morphostatic c e l l s of l i n e s 10-la and 19-2b  91  Micronuclear segregation at c e l l d i v i s i o n .  93  Number of m i c r o n u c l e i at d i v i s i o n i n vi  \  vii Table  Page c e l l s of d i f f e r e n t ages  97  XV". Number of m i c r o n u c l e i a t d i v i s i o n i n c e l l s of d i f f e r e n t ages XVI.  V a r i a t i o n i n t h e number of n u c l e i i n exautogamoussfirst c e l l cycle d i v i d e r s  X V I I . Comparison of v a r i a b l e s f o r t h e o f MR by heat shock  98 . . .  99  induction 158  \  LIST OF  FIGURES  Figure 1.  Page  S e c t i o n through the I5etraurelia  cortex '  of Paramecium  •  •  3  2.  I n t e r n a l morphology of P_. t e t r a u r e l i a  3.  Nuclear events during morphogenesis  prefission  4.  Nuclear events d u r i n g  the  5.  Nuclear events during  autogamy  6.  I s o l a t i o n scheme of the i n v o l v i n g a tarn gene  7.  P r o b i t a n a l y s i s _ o f tarn A c e l l c y c l e l e n g t h s .  49  8.  A comparison of c e l l morphology  52  9.  M i s s e g r e g a t i o n of the macronucleus at fission  10. 11.  13.  14.  10  sexual process  . .  F-^progeny i n a  cross  A comparison of macronuclear p r e f i s s i o n morphogenesis Penetrance of the tarn A and  am  32  .  57  62  The frequency of complete macronuclear m i s s e g r e g a t i o n i n tarn A/tam A and am/am c e l l s as a f u n c t i o n of c l o n a l age experiment comparing the i n r e l a t e d p a i r s of  64 number  T h e o r e t i c a l diagram deomonstrating how l a r g e numbers of m i c r o n u c l e i may a r i s e i n c e l l s e x p r e s s i n g the tarn phenotype . . . . viii  54  genes as  of c l o n a l age  Diagram of the of m i c r o n u c l e i sister cells  14 17  i n c e l l s e x p r e s s i n g the tarn phenotype  a function 12.  7  . . . .  70  73  ix Figure  page  1$. A comparison of e a r l y , middle and l a t e d i v i d e r s  75  16. I l l u s t r a t i o n of the d i f f e r e n c e i n m i c r o n u c l e a r placement i n ±/± and tarn A/tam A d i v i d i n g c e l l s  77  17. The d i f f e r e n c e i n t r i c h o c y s t morphogenesis  80  18. The experiment comparing the frequency of macronuclear m i s s e g r e g a t i o n a t the f i r s t and second exautogamous d i v i s i o n 19.  The e f f e c t  . . . .  $4  o f age on the frequency  of m i c r o n u c l e a r m i s s e g r e g a t i o n 20. Abnormal mating c o n f i g u r a t i o n s from +/+ X 21-3c  95 103  21. Formation o f the pw A/pw A ; t s l ^ t s l ( m a c . ) +  t s l / t s l + / p w A+/pwAA+(mic.) heterokaryon . . . .  153  LIST OF PLATES Plate  page  " 1. V a r i o u s a s p e c t s of macronuclear misplacement i n d i v i d i n g tarn A homozygotes. . . . 2. A comparison of complete and macronuclear m i s s e g r e g a t i o n  partial  163  3. V e g e t a t i v e tam A and tarn G homozygotes . . . . 4 . Improper placement of the d i v i d i n g macronucleus i n tam A/tam A c e l l s , g i v i n g r i s e to 'fragments' of the macronucleus at the completion of c e l l and macronuclear d i v i s i o n  161  165  .  5 . Amacronucleate v e g e t a t i v e c e l l s of tam A homozygotes showing i r r e g u l a r numbers of micronuclei  167  169  6o  6.  V e g e t a t i v e phase of the l i f e c y c l e showing i r r e g u l a r d i s t r i b u t i o n of m i c r o n u c l e i at f i s s i o n i n tam A homozygotes  171  Exautogamous tam A/tam A c e l l s j u s t complete i n g the f i r s t postautogamous c e l l c y c l e . . .  173  8. Exautogamous tam G/tam G c e l l s j u s t completi n g the f i r s t postautogamous c e l l c y c l e . . .  175  7.  9. The completion of the f i r s t  cell  cycle  f o l l o w i n g autogamy i n tam A/tam A c e l l s . . . .  177  10. Exautogamous c e l l s from l i n e 21-3c  179  11. Exautogamous c e l l s from l i n e 21-3c 12. 21-3c conjugants showing l a c k of n u c l e a r synchrony w i t h pw A/pw A p a r t n e r s  181 183  13. Cytoplasmic i n c l u s i o n s c h a r a c t e r i s t i c of sp homozygotes. . . . . . . .  185  x  ACKNOWLEDGMENT  The author wishes t o thank Dr. J.D. Berger f o r h i s s u p e r v i s i o n of t h i s study and h e l p f u l c r i t i s m o f t h e f o l l o w i n g manuscript.  S p e c i a l thanks go t o my husband,  Glenn Morton, f o r h i s h e l p w i t h the computing photography,  and t h e  f o r h i s understanding and encouragement,  and f o r h i s u n f a i l i n g moral and f i n a n c i a l support. Thanks a r e a l s o warmly extended t o C a r o l P o l l a c k and Don Jones f o r t h e i r many i n s p i r i n g d i s c u s s i o n s a t seminars (and wholenars) special  and i n the l a b o r a t o r y , and f o r t h e i r very  friendship.  - T h i s r e s e a r c h was supported by N.R.C. o p e r a t i n g grant A67-6300 t o Dr. J.D. Berger.  xi  INTRODUCTION  The i n i t i a l aim of t h i s study was t o analyse the r e v e r s i b l e i n t e r a c t i o n between the p o s t z y g o t i c macronucleus and the p r e z y g o t i c macronuclear fragments i n Paramecium t e t r a u r e l i a  (Sonneborn, 1975)  exconjugants.  T h i s i n t e r a c t i o n r e s u l t s i n s e l e c t i v e s u p p r e s s i o n of s y n t h e s i s i n macronuclear fragments when a new,  DNA  immature  macronucleus i s present (Berger, 1973a).  I t was hoped t h a t the nature of the i n t e r a c t i o n between fragments and anlagen could be e l u c i d a t e d through the s e l e c t i o n and a n a l y s i s of induced mutations which a l t e r e d the normal suppressive e f f e c t of macronuclear anlagen on s y n t h e s i s i n macronuclear fragments.  I t was  DNA  expected t h a t  i n such mutant c e l l s macronuclear r e g e n e r a t i o n would occur i n the presence of macronuclear anlagen;system was  The  selection  s u c c e s s f u l and t h r e e p u t a t i v e mutants which  underwent macronuclear r e g e n e r a t i o n were r e c o v e r e d from a t o t a l of 8,100  i n d i v i d u a l l y screened c e l l s .  Early analysis  of two of the v a r i a n t s (tarn A and tarn G) r e v e a l e d that the cause of macronuclear r e g e n e r a t i o n i n exautogamont was  cells  due not t o the expected a l t e r a t i o n i n the i n t e r a c t i o n  between macronuclear anlagen and p r e z y g o t i c macronuclear  2 fragments but t o a l t e r a t i o n s i n n u c l e a r behaviour at d i v i s i o n , which stem from improper cytoplasmic of the p r e d i v i s i o n n u c l e i .  localization  A n a l y s i s of the t h i r d v a r i a n t  r e v e a l e d abnormal behaviour of the m i c r o n u c l e i the sexual  cell  during  process.  In view of the f i n d i n g s , t h i s study focused a n a l y s i s of the three v a r i a n t s recovered soguanidine mutagenesis.  upon the  following nitro-  I t i s hoped that a f u r t h e r under-  standing of n u c l e a r morphogenesis at c e l l d i v i s i o n and n u c l e a r r e o r g a n i z a t i o n w i l l be gained obtained.  from the r e s u l t s  Before c o n t i n u i n g with the a n a l y s i s some back-  ground i n f o r m a t i o n about the organism and w i l l be  at  it's life  cycle  presented.  C e l l Cortex The h o l o t r i c h o u s c i l i a t e ,  Paramecium t e t r a u r e l i a , i s  a s i n g l e - c e l l e d animacule approximately 115;um by 41  .um  (1)  at  i n t e r f i s s i o n age  0.6  V  ;  (Kaneda and Hanson, 1976). The  c e l l c o r t e x c o n t a i n s l o n g i t u d i n a l rows of c o r t i c a l u n i t s known as k i n e t i e s .  Each u n i t c o n s i s t s of one  and b a s a l bodies (kinetosomes), a perisomal filament  or two  cilia  sac and a long  (kinetodesma) t h a t runs p a r a l l e l t o the  kinety  from the s i d e of the p o s t e r i o r b a s a l body ( F i g u r e 1).  The  (1) In t h i s t h e s i s a l l c e l l c y c l e stages are i n d i c a t e d by decimal f r a c t i o n s which express the r e l a t i v e p o s i t i o n of the c e l l i n the c e l l c y c l e .  F i g u r e 1.  S e c t i o n through the c o r t e x of P. ( A f t e r Ehret an'd Powers,  1959)  tetraurelia.  / /  4  FIGURE 1  5  a n t e r i o r and p o s t e r i o r boundaries of each c o r t i c a l u n i t are d e f i n e d by t r i c h o c y s t s , one  at each boundary.  t r i c h o c y s t s are ejectosomes between two  The  and f i v e micrometers  i n l e n g t h , a l i g n e d j u s t beneath the c e l l ' s s u r f a c e perpendicular  t o the plasma membrane.  They are  and  surrounded  by a s i n g l e membrane and are composed of three p a r t s : body, the t i p and the  sheath.  the  Both the body and the t i p  of the t r i c h o c y s t are composed of a t r y p s i n - d i g e s t i b l e p r o t e i n and. the sheath i s composed of a p e p s i n - d i g e s t i b l e p r o t e i n and  g l y c o p r o t e i n (Esteve, 1 9 7 4 ) .  In the mature  t r i c h o c y s t , the p r o t e i n i s arranged i n a c r y s t a l l i n e s t r u c t u r e w i t h a p e r i o d i c i t y of 7 - l 6 n m ( P o l l a c k , 1 9 7 4 ) .  The  s t r u c t u r e of t r i c h o c y s t s changes d r a m a t i c a l l y upon e x t r u s i o n .  According  t o Kaneda and Hanson (1974) the c e l l  can be d i v i d e d i n t o two cludes the upper l e f t  zones:  surface  the adoa?al zone (which i n -  quadrant, the o r a l groove and  the  v e s t i b u l e ) r e l a t e d almost e n t i r e l y t o f e e d i n g , and the zone (which i n c l u d e s the remaining c e l l s u r f a c e ) with m o t i l i t y . tral  The  cytoproct  concerned  (anus) i s l o c a t e d on the'ven-  ( o r a l ) s u r f a c e i n the p o s t e r i o r p a r t of the c e l l .  c o n t r a c t i l e vacuole  anoral  pores are found on the d o r s a l  Two  (aboral)  s u r f a c e l y i n g i n the i n t e r k i n e t y spaces, one  a n t e r i o r and  the other p o s t e r i o r .  i s about  Each of these vacuoles  t h i r d of the d i s t a n c e from one  end  of the  cell.  one  6 Nuclei Paramecium t e t r a u r e l i a possess two The w i l d type  c e l l c o n t a i n s two,  germinal m i c r o n u c l e i and  one  macronucleus ( F i g u r e 2 ) .  types  of n u c l e i .  s m a l l , round, d i p l o i d ,  l a r g e r , polygenomic, somatic  During  v e g e t a t i v e growth the  macronucleus i s r e s p o n s i b l e f o r the b i o s y n t h e t i c events which d i r e c t RNA  c e l l phenotype and  s y n t h e s i s and  amount of RNA 1967)..  output  s y n t h e s i z e d by the m i c r o n u c l e i  i n t e r f i s s i o n age,  macronucleus i s round, and of the b u c c a l c a v i t y .  Kaneda and Hanson, 1974) l i e s adjacent  the  t o the d o r s a l s i d e  S t r u c t u r a l l y , the macronucleus i s  (Jurand,  D i p p e l l and S i n t o n , 1963;  s m a l l bodies have a DNA  chromatin  Beale  and  Stevenson and  Lloyd,  1971).  e q u i v a l e n t of about one - chromosome  and they are i n t e r c o n n e c t e d by very f i n e DNA 1967  (Pasternak,  I t c o n t a i n s many s m a l l , electron-dense  Young, 1962;  small  p o r t i o n of the c e l l c y c l e  bodies and numerous, l a r g e r n u c l e o l i  The  Macronuclear  i s c o n s i d e r a b l e and masks the  During the morphostatic  (0.1-0.7,  complex.  c e l l function.  threads  (Wolfe,  i n Paramecium t e t r a u r e l i a and Tetrahymena p y r i f o r m i s ;  Morat, 1973  i n Colpidium  campylum).  macronucleus i s approximately genomes (Woodard, et a l . , 1961; Berger, 1973b; Morton, 1 9 7 4 ) .  The  DNA  content  e q u i v a l e n t t o 860 A l l e n and Gibson,  of a  haploid 1972;  Macronuclear S phase has  r e p o r t e d t o begin a t 0 . 2 5 of the c e l l c y c l e , and  been  continues  up t o k a r y o k i n e s i s (Kimball, et a l . , I 9 6 0 ; Woodard, et a l . , 1961;  Berger, 1 9 7 1 ) .  There i s no G  9  p e r i o d i n P_. t e t r a u r e l i a .  FIGURE 2.  Internal  morphology  of P.  cv, c o n t r a c t i l e  tetraurelia.  ci,  cilia;  ma,  macronucleus; mi, m i c r o n u c l e i ;  og, o r a l groove; f v , food tr,  trichocysts.  vacuole;  vacuole;  8  I n t e r p r e t a t i o n s o f t h e o r g a n i z a t i o n o f the macronucleus i n members of the Oligohymenophora  are v a r i e d .  There have  been s e v e r a l c o n j e c t u r e s r a n g i n g from random r e p l i c a t i o n and assortment of unordered chromosomes t o the macronucleus being made up of complete genome, d i p l o i d s u b u n i t s (see Nyberg,  1976).  Probably the most accepted v e r s i o n of macronuclear  o r g a n i z a t i o n i s t h a t the macronucleus i s made up of h a p l o i d subunits c o n t a i n i n g complete genomes. to  be t r u e i n Tetrahymena  This at least  (see O r i a s and F l a c k s ,  appears  1975).  D i v i s i o n o f the, macronucleus i s a m i t o t i c , t h a t i s , without condensation of chromosomes or the f o r m a t i o n o f a t y p i c a l mitotic spindle.  At about i n t e r f i s s i o n age 0.7  the  macronucleus s h i f t s from i t s i n t e r f i s s i o n l o c a t i o n on the d o r s a l s i d e o f the g u l l e t and moves t o the centre p a r t of the  cell.  (Kaneda and Hanson,  1974).  In t h i s p o s i t i o n t h e '  macronucleus begins t o s w e l l but m a i n t a i n s i t s s p h e r i c a l , shape. to  At about i n t e r f i s s i o n age 0.85  the macronucleus begins  elongate very s l i g h t l y and migrates towards a s u b c o r t i c a l  l o c a t i o n on the d o r s a l aspect, of the c e l l  (Figure 3).  Simult-  aneously, m i c r o t u b u l e s appear i n the cytoplasm p a r a l l e l t o the  n u c l e a r envelope but without connection t o i t (Stevenson  and L l o y d , the  1971).  R e p o s i t i o n e d i n the s u b c o r t i c a l r e g i o n ,  macronucleus e l o n g a t e s and, a t the same time, the e x t r a -  n u c l e a r m i c r o t u b u l e s grow a l o n g the l o n g i t u d i n a l a x i s o f the  Figure 3 .  Nuclear events d u r i n g ppa?edivision morphogenesis., fv,  food vacuoles;  mi, m i c r o n u c l e i . of the c e l l .  ma, macronucleus; 0.7 -  1 . 0 , i n t e r f i s s i o n age  At 0 . 7 the macronucleus moves  away from the d o r s a l s i d e of the b u c c a l c a v i t y and assumes a new l o c a t i o n i n the centre of the c e l l .  At age 0 . 8 the macronucleus begins  e l o n g a t i o n and r e p o s i t i o n s i n a s u b c o r t i c a l l o c a t i o n on the d o r s a l s i d e of the c e l l .  At  age 0 . 8 5 the m i c r o n u c l e i have completed m i t o s i s and the macronucleus has elongated.  Con-  s t r i c t i o n o f the macronucleus occurs a t about age 0 . 9 and c e l l d i v i s i o n i s completed a t age  1.0.  ma  0.7  0.8  FIGURE 3  0-85  0.9  12 cell.  When e l o n g a t i o n commences, i n t r a n u c l e a r m i c r o t u b u l e s  appear.  Between i n t e r f i s s i o n age 0.8$  -  0.9  the macronucleus  i  becomes a t h i n rod forming a 'backbone' along the d o r s a l c o r t e x of the c e l l .  Simultaneously, the i n t r a n u c l e a r micro- -  t u b u l e s a l i g n along the l o n g i t u d i n a l a x i s of the nucleus the e x t r a n u c l e a r m i c r o t u b u l e s become reduced  and  i n number.  D u r i n g . c o n s t r i c t i o n of the macronucleus, a l a r g e number of m i c r o t u b u l e s appear i n p a r a l l e l arrangements a l o n g the l o n g i t u d i n a l a x i s of the d i v i d i n g macronucleus i n the region.  isthmus  K a r y o k i n e s i s and c y t o k i n e s i s occur c o n c u r r e n t l y .  Upon completion of f i s s i o n the daughter macronuclei c o n t r a c t i n t o the t y p i c a l o v a l form of the i n t e r f i s s i o n c e l l  and  migrate back t o the i n t e r f i s s i o n p o s i t i o n at the d o r s a l s i d e of the g u l l e t .  Recently, Inaba and Kudo (1972) found t h a t  i n P. multimicronucleatum  the e x t r a n u c l e a r m i c r o t u b u l e s ,  which arrange j u s t p r i o r t o c e l l d i v i s i o n , do connect pores of the macronuclear  with  and m i c r o n u c l e a r envelopes.  At  the completion of macronuclear  d i v i s i o n no t r a c e of the  n u c l e a r m i c r o t u b u l e s remains.  I t i s not c l e a r what r o l e  intrathe  i n t a - and e x t r a n u c l e a r m i c r o t u b u l e s p l a y d u r i n g k a r y o k i n e s i s . I t might be suggested t h a t the i n t r a n u c l e a r m i c r o t u b u l e s  may  p a r t i c i p a t e i n chromosome movement d u r i n g n u c l e a r d i v i s i o n . However, attachments  between these m i c r o t u b u l e s and  bodies have not been observed.  chromatin  I t i s g e n e r a l l y assumed t h a t  the m i c r o t u b u l e s are i n v o l v e d i n n u c l e a r e l o n g a t i o n (e.g. Inaba and Kudo, 1 9 7 2 ) .  13  The m i c r o n u c l e i l i e i n s m a l l depressions of the macronucleus.  on the  surface  They are s m a l l , round n u c l e i surrounded  by a n u c l e a r envelope t h a t remains i n t a c t d u r i n g m i t o s i s .  The micronuclear  S phase begins about 50^ of the  through the c e l l c y c l e and (Pasternak,  l a s t s f o r about 80 minutes  D i v i s i o n of the m i c r o n u c l e i  1967).  about i n t e r f i s s i o n age  0.85  and  intranuclear mitotic spindle  The  way  occurs  i s c h a r a c t e r i z e d by  (Stevenson and  Lloyd,  at  an 1971).  L i f e Cycle of Paramecium t e t r a u r e l i a The mean c e l l c y c l e l e n g t h under normal c u l t u r e con-  d i t i o n s ( 2 7 ° C and w e l l fed) i s approximately 5.00  hours f o r  w i l d type c e l l s .  cycle  The  v e g e t a t i v e p a r t of the l i f e  l a s t f o r s e v e r a l hundred c e l l g e n e r a t i o n s . u a l l y age  and  die unless meiosis,  a t i o n of a new place.  The  cells  event-  f e r t i l i z a t i o n and the  macronucleus (nuclear r e o r g a n i z a t i o n )  may  form-  takes  Three types of n u c l e a r r e o r g a n i z a t i o n occur i n P.  tetraurelia: 1 . Conjugation  (Figure 4 )  C e l l s of opposite mating type (Sonneborn, 1 9 3 7 ) form c o u p l e t s by j o i n i n g at t h e i r o r a l s u r f a c e . undergo meiosis  and  The  give r i s e to e i g h t h a p l o i d  Seven of these degenerate and  micronuclei products.  one migrates t o the p a r o r a l  cone (a unique s t r u c t u r e which forms near the mouth d u r i n g  F i g u r e 4.  Nuclear events d u r i n g the sexual process ( c o n j u g a t i o n ) i n P. t e t r a u r e l i a . f v , food vacuoles; ma, macronucleus; mi, m i c r o n u c l e i . a. m i c r o n u c l e i i n e a r l y prophase of the f i r s t pregamic d i v i s i o n ; b. m i c r o n u c l e i i n the t y p i c a l c r e s c e n t stage of the f i r s t pregamic d i v i s i o n ; c. e l o n g a t i o n and e a r l y fragmentation i n the macronucleus, completion of the f i r s t pregamic d i v i s i o n ; d. s k e i n f o r m a t i o n i n the macronucleus, completion of the second pregamic d i v i s i o n ; e. macronuclear fragmentation, f o r m a t i o n of the isogamic n u c l e i (migratory and stationary); f. r e c i p r o c a l exchange of the male pronucleus; g. f o r m a t i o n of the z y g o t i c nucleus (synkaryon); h. completion of the second m i t o t i c d i v i s i o n of the synkaryon; i. u n c o u p l i n g of the exconjugants, d i f f e r e n t i a t i o n of the synkaryon products; j. s e g r e g a t i o n of the two new macronuclei (anlagen) at the f i r s t c e l l d i v i s i o n . "  15  FIGURE 4  16 m e i o s i s ) where i t completes  a m i t o t i c d i v i s i o n t o give r i s e  t o one s t a t i o n a r y and one m i g r a t o r y isogametic p r o n u c l e i . R e c i p r o c a l exchange of the migratory p r o n u c l e i takes p l a c e between the c e l l s .  F u s i o n of the exchanged n u c l e i w i t h the  indigenous s t a t i o n a r y n u c l e i occurs t o form d i p l o i d , nuclei  (synkaryon) i n both c e l l s .  twice, m i t o t i c a l l y .  The synkaryon  zygotic  divides  In each c e l l two products become macro-  n u c l e a r anlagen and two become the new  micronuclei.  The  v e g e t a t i v e n u c l e a r complement i s r e s t o r e d at the f i r s t d i v i s i o n when the anlagen are segregated t o the cells.  cell  daughter  During c o n j u g a t i o n , the macronucleus breaks down i n t o  fragments  (about 3 5 / c e l l ) .  segregated t o daughter resumed.  DNA  These fragments  are randomly  c e l l s when the v e g e t a t i v e c y c l e i s  s y n t h e s i s i s suppressed i n the fragments  and  e v e n t u a l l y they are a u t o l y s e d (Berger, 1973a).  2. Autogamy ( F i g u r e 5) Autogamy t a k e s p l a c e i n uncoupled form of ' s e l f i n g ' .  cells.  It i s a  M i c r o n u c l e a r events proceed as d e s c r i b e d  f o r c o n j u g a t i o n , but the isogametic p r o n u c l e i fuse  immediately  a f t e r t h e i r f o r m a t i o n , f o r e g o i n g any n u c l e a r exchange w i t h another c e l l . synkaryon  Since the p r o n u c l e i which fuse t o form the  i n an autogamous c e l l are s i s t e r m i t o t i c  products  of a s i n g l e h a p l o i d n u c l e u s , the synkaryon i s completely homozygous.  Figure 5 .  Nuclear events d u r i n g autogamy i n P. tetraurelia. f v , food vacuole; ma, macronucleus; mi, m i c r o n u c l e u s . a. m i c r o n u c l e i .in e a r l y prophase of the f i r s t pregamic d i v i s i o n ; b. c r e s c e n t stage of the f i r s t pregamic division; c. completion of the, f i r s t pregamic division; d. s k e i n f o r m a t i o n of the macronucleus, completion of the second pregamic d i v i s i o n of the m i c r o n u c l e i ; e. f r a g m e n t a t i o n of the macronucleus, f o r m a t i o n o f the isogametic p r o n u c l e i ; f. f u s i o n of the p r o n u c l e i t o form the synkaryon; g. and h. f i r s t and second m i t o t i c d i v i s i o n o f the synkaryon; i . and j . d i f f e r e n t i a t i o n of the synkaryon products; k. s e g r e g a t i o n of the macronuclear anlagen at t h e f i r s t c e l l d i v i s i o n ; 1. v e g e t a t i v e c y c l e resumed.  FIGURE 5  19 3. Cytogamy Cytogamy does not appear t o be a phenomenon i n i t s own r i g h t , but i s , r a t h e r , a f a i l u r e of the c e l l s t o complete c o n j u g a t i o n .  Cytogamy can only take  place  d u r i n g c o n j u g a t i o n , s i n c e i t i s e s s e n t u a l l y 'autogamy a t conjugation'.  Coupled c e l l s f o l l o w the same n u c l e a r  events  a l r e a d y d e s c r i b e d f o r c o n j u g a t i o n with the e x c e p t i o n t h a t exchange of the migratory p r o n u c l e i does'not take p l a c e . C e l l s t h a t complete t h i s completely  homozygous.  ' f a l s e c o n j u g a t i o n ' are  20  MATERIALS AND METHODS  Culturing  Techniques  C e l l s were grown e i t h e r i n c e r o p h y l l or i n grass media (pH6.8 - 7.0) w i t h A e r o b a c t e r aerogenes as the food (Sonneborn, fed  1970).  every two weeks.  temperature  organism  Stock c u l t u r e s were kept a t 17°C and A l l experiments were conducted 'at room  u n l e s s otherwise  specified.  C u l t u r e Media c e r o p h y l l or grass powder  12.5  gms  Na HP0^  3.75  gms  H0  5,000 mis  2  2  (distilled)  The c e r o p h y l l ( g r a s s ) and b u f f e r were added t o f i v e l i t e r s of d i s t i l l e d water and b o i l e d f o r f i v e minutes. c u l t u r e f l u i d was, then b o t t l e d and a u t o c l a v e d .  The  The c u l t u r e  f l u i d was i n o c u l a t e d w i t h the food organism 21+ hours b e f o r e use..  The pH was a d j u s t e d immediately b e f o r e use.  Stocks —  — —  —•—~•  The v a r i o u s s t r a i n s of Paramecium t e t r a u r e l i a i n t h i s study are d e s c r i b e d i n Table 1.  \  used  21  TABLE I STOCKS USED IN THE COURSE OF THIS STUDY  STOCK  GENE  51.S  REMARKS AND REFERENCES Wild-type stock  (Sonneborn,  1975)  d4-43  am, nd6  Both r e c e s s i v e mutations were r e c o v e r e d a f t e r UV mutagenesis (Sonneborn, 1954) and c h a r a c t e r i z e d by N o b i l i (1959, 1961). D4-43 i s d e r i v e d from stock d 4 - 2 . The am gene causes m i s s e g r e g a t i o n of the macronucleus at c e l l d i v i s i o n and miss e g r e g a t i o n of anlagen a t the f i r s t and/ or second exconjugant (or exautogamont) cell division. The am gene a l s o causes m i s s e g r e g a t i o n of m i c r o n u c l e i a t c e l l division. The nd6 gene, when homozygous, causes f a i l u r e of t r i c h o c y s t s t o d i s charge when t h e c e l l i s s t i m u l a t e d w i t h an i r r i t a n t ( p i c r i c a c i d ) .  d4-ll6  ftA  T h i s r e c e s s i v e mutation was r e c o v e r e d a f t e r X-ray treatment ( P o l l a c k , 1970). The mutation causes incomplete morphogenesis of t r i c h o c y s t s . The t r i c h o c y s t s are unattached t o the c e l l ' s c o r t e x , are t i p l e s s , a r e f o o t b a l l shaped, and do not d i s c h a r g e ( P o l l a c k , 1970).  d4-103  ftB  T h i s r e c e s s i v e mutation was r e c o v e r e d a f t e r treatment of 51.S c e l l s w i t h NTG. The phenotype i s s i m i l a r t o t h a t of f t A but i s not a l l e l i c w i t h f t A  '  (Pollack7~T970).  d4-94  pwA  T h i s r e c e s s i v e mutation was r e c o v e r e d a f t e r NTG mutagenesis of stock 51.S c e l l s . Homozygous pwA c e l l s - d o not a v o i d i n s o l u t i o n s higE i n Na+ K or Ba*(Kung, 1971).  d4-106  stA  T h i s r e c e s s i v e mutation was r e c o v e r e d a f t e r NTG treatment of s t o c k 51.S c e l l s . The gene was c h a r a c t e r i z e d by P o l l a c k (1970, 1974). When homozygous i t causes incomplete morphogenesis of t r i c h o c y s t s . T r i c h o c y s t s a r e stubby i n shape and are u s u a l l y tipl:Css3s or have skewed t i p s . Some t r i c h o c y s t s are a t t a c h e d t o the c e l l ' s c o r t e x and can d i s c h a r g e . Most of them are unattached.  22 STOCK  d4-107  GENE  stB  REMARKS AND REFERENCES This_recessive,mutation i s s i m i l a r i n phenotype t o stA with the exception t h a t stB does not show/a w i l d - t y p e phenotype under m i l d s t a r v a t i o n as stA does.  d4-1030  tamA  T h i s r e c e s s i v e mutation was recovered a f t e r NTG mutagenesis of stock 5 1 . s c e l l s . Tam A .causes missegregation of n u c l e i a t cell division. I t a l s o causes improper t r i c h o c y s t morphogenesis. The t r i c h o c y s t s a r e f o o t b a l l shaped, t i p l e s s and unattached t o the c o r t e x . They do not discharge when s t i m u l a t e d w i t h p i c r i c a c i d ,  d4-1031  tamG  T h i s r e c e s s i v e mutation was recovered a f t e r NTG mutagenesis of stock 5 1 . s c e l l s . Tam G has a s i m i l a r phenotype t o tam A. In a d d i t i o n t o the e f f e c t on n u c l e a r miss e g r e g a t i o n and t r i c h o c y s t morphogenesis, tam G c e l l s f r e q u e n t l y have e x t r a n u c l e i at n u c l e a r r e o r g a n i z a t i o n . Trichocysts are stubby.  d4-108  tam8  T h i s r e c e s s i v e mutation was recovered from NTG t r e a t e d stock 5 1 . s c e l l s (Beisson and R o s s i g n o l , 1 9 7 5 ) . The mutation causes missegregation o f n u c l e i a t c e l l . d i v i s i o n and f a i l u r e o f t r i c h o c y s t s t o d i s c h a r g e . The t r i c h o c y s t s i n tam/tam c e l l s a r e not a l i g n e d and are unattached (Sonneborn, 1974; Beisson and R o s s i g n o l , 1975 and Ruiz et a l . , 1 9 7 6 ) .  tam38  T h i s r e c e s s i v e mutation was recovered from UV t r e a t e d stock d 4 - 2 c e l l s (Adoutte and Beisson, 1 9 7 0 ) . When homozygous, tam 38 causes n u c l e a r m i s s e g r e g a t i o n a t c e l l d i v i s i o n ; f o o t b a l l shaped, n o n d i s charge t r i c h o c y s t s and e x t r a n u c l e i a t n u c l e a r r e o r g a n i z a t i o n . Tam 38 i s very s i m i l a r t o tam Gj (except f o r the shape of t h e t r i c h o c y s t s ) and tam A.  t-33  T h i s r e c e s s i v e mutation a l s o causes the tam phenotype when homozygous. T - 3 3 was recovered from NTG t r e a t e d stock d 4 - 2 cells.  23 STOCK  GENE  d.4-108  tam21  d4-85  tslll  REMARKS AND REFERENCES  Tam 21 i s a temperature-sensitive, tam  mutation recovered a f t e r treatment of stock d4-2 c e l l s w i t h NTG (Beisson and R o s s i g n o l , 1975). T r i c h o c y s t s a r e f o o t b a l l shaped, unattached and f a i l t o d i s charge when the c e l l i s s t i m u l a t e d w i t h picric acid. The growth r a t e ' i s slow (2-3 f i s s i o n s p e r day) and the c e l l s d i e w i t h i n 36 hours o f b e i n g r a i s e d t o the r e s t r i c t i v e temperature (35°C). This i s a temperature-sensitive, recessive, l e t h a l mutation. I t was r e c o v e r e d a f t e r treatment of d4-2 c e l l s w i t h UV. Tslll/ t s l l l w i l l d i e w i t h i n 48 hours a f t e r being r a i s e d t o the r e s t r i c t i v e temperature  (Beisson and R o s s i g n o l , 1969).  24  Mutagenesis P o p u l a t i o n s o f stock 5 1 . s c e l l s to  about  one m i l l i o n c e l l s .  by m i l d c e n t r i f u g a t i o n in  100  ( w i l d type) were expanded  The c e l l s were then c o n c e n t r a t e d  ( 1 . 0 min a t 1 0 0 0 rpm) and resuspended  mis of D r y l ' s s o l u t i o n  (Dryl  1959).  agen N-methyl-N'-nitro-N-nitrosoguanidine  The chemical mut(NTG,CH^.N(N0).C(NH).  NH.NOg, Sigma chemicals) was d i s s o l v e d i n 1 0 0 mis o f D r y l ' s s o l u t i o n t o a c o n c e n t r a t i o n of 7 5 ug/ml (Kung 1 9 7 1 , 1975).  Peterson,  The c o n c e n t r a t e d c e l l suspension was mixed w i t h the  d i s s o l v e d NTG and allowed t o stand f o r 5 0 t o 6 0 minutes. A f t e r treatment the c e l l s were washed t h r e e times i n s t e r i l e D r y l ' s s o l u t i o n and suspended i n f r e s h c u l t u r e f l u i d w i t h enough b a c t e r i a t o a l l o w f o r t h r e e t o f o u r d o u b l i n g s o f t h e c e l l population.  T h i s c u l t u r e was maintained f o r 2 4 t o 4 8  hours t o a l l o w f o r the exhaustion of t h e food and t h e occurrence o f autogamy.  Precautions D i l u t e n i t r i c a c i d was a v a i l a b l e at a l l times d u r i n g mutagenesis.  A l (Contaminated  u t e n s i l s and glassware were  p l a c e d i n d i l u t e n i t r i c a c i d c o n t a i n e r s where they were l e f t for  one week i n t h e l i g h t .  Mutagenesis  p l e x i g l a s s safebox i n a fumehood.  was conducted  ina  25 Population"Counts P o p u l a t i o n counts were made by s e r i a l d i l u t i o n of 1.0 ml o f the c e l l c u l t u r e .  The average was determined  from d i r e c t counts of t h r e e or more samples.  Test f o r Autogamy  1  '  Between 30-50 c e l l s were p l a c e d i n a drop o f D r y l ' s s o l u t i o n on a c l e a n microscope s l i d e . stain  A drop o f D i p p e l l  ( D i p p e l l , 1955) was added t o the drop o f c e l l s and  covered w i t h a c o v e r s l i p . low power (20x) o b j e c t i v e .  O b s e r v a t i o n s were made u s i n g a The cytoplasm s t a i n e d green  i n c o n t r a s t t o the n u c l e i which s t a i n e d dark brown.  Cells  i n autogamy c o n t a i n e d many macronuclear fragments  rather  than t h e compact nucleus seen i n v e g e t a t i v e c e l l s .  The  lowest l e v e l that was a c c e p t a b l e was 95$ autogamy.  Determination of V e g e t a t i v e C e l l Death Immediately a f t e r mutagenesis,  200 c e l l i s o l a t e s , both  from the mutagen t r e a t e d c e l l p o p u l a t i o n and from an u n t r e a t e d c o n t r o l p o p u l a t i o n , were made and allowed t o grow f o r two days. i  The frequency of v e g e t a t i v e c e l l death was a s c e r t a i n e d . Clones which c o n t a i n e d no more than f o u r c e l l s were scored as dead (normal l i n e s had s e v e r a l hundred c e l l s ) .  The frequency of  v e g e t a t i v e death gave an i n d i c a t i o n of the l e v e l of t o x i c i t y of NTG and t h i s percentage was then s u b t r a c t e d from t h e p e r -  , centage of exautogamous death.  26  The percentage of death i n  t h e . c o n t r o l i s o l a t e s i n d i c a t e d the l e v e l o f t o x i c i t y solution.  Dryl's solution i s a p h y s i o l o g i c a l s a l t  which can be t o x i c i f the c e l l s are l e f t  of.Dryl's  solution  i n i t too long.  Determination of Exautogamous Death F o l l o w i n g the f i r s t autogamy a f t e r mutagenesis,  200  exautogamonts were i s o l a t e d and allowed t o grow f o r two. t o t h r e e days. dead.  Clones w i t h f o u r c e l l s or fewer were s c o r e d as  S i n c e autogamy r e s u l t s i n complete homozygosis  of the  genome, the frequency of exautogamous death g i v e s an i n d i c a t i o n o f the r e l a t i v e frequency of l e t h a l mutations  produced  by t h e mutagen and i s thus an i n d i c a t i o n of the e f f e c t i v e n e s s of  the mutagenic  treatment.  Mutagenic  exautogamous death l e v e l of below 45%  experiments w i t h an were c o n s i d e r e d unsuc-  c e s s f u l and t h e r e f o r e d i s c a r d e d .  Method of S e l e c t i o n Mutagen t r e a t e d exautogamous c e l l s were p l a c e d i n p e t r i d i s h c u l t u r e s and f e d .  E a r l y d i v i d e r s were s e l e c t e d from the  " p e t r i d i s h c u l t u r e s and were i s o l a t e d i n t o d e p r e s s i o n s l i d e wells.  S i s t e r c e l l s were allowed t o separate and one s i s t e r  c e l l from each of the i s o l a t e s was d r i e d on albumin-coated s l i d e s i n rows.  1969),  The s l i d e s were s t a i n e d w i t h Azure A (Berger,  and the c e l l s were scored f o r the presence of regener-  27 a t i n g macronuclear fragments and f o r abnormal numbers of m i c r o n u c l e i and macronuclear anlagen.  The l i v i n g  sister  c e l l s of s t a i n e d c e l l s w i t h abnormal n u c l e a r complements were r e t a i n e d as p u t a t i v e  mutants.  Test f o r T r i c h o c y s t Discharge A s m a l l sample o f l i v e c e l l s was p l a c e d i n a drop of D r y l ' s s o l u t i o n on a microscope s l i d e . picric  a c i d was added.  A drop o f s a t u r a t e d  Wild-type c e l l s d i s c h a r g e t h e i r  e n t i r e complement of t r i c h o c y s t s w i t h i n a second.  Cells  homozygous f o r tarn, jst, f t and nd f a i l t o d i s c h a r g e t r i c h o cysts.  Discharged t r i c h o c y s t s appear as a h a l o of t i n y  h a i r s when viewed w i t h a low power o b j e c t i v e .  Test f o r t h e Presence o f T r i c h o c y s t s One or two c e l l s were p l a c e d i n a minute drop o f D r y l ' s s o l u t i o n on a microscope s l i d e .  A cover s l i p was added  immediately and pressed down on the s l i d e cells. trast  t o r u p t u r e the  The c e l l s were examined w i t h a n e g a t i v e phase cono i l immersion  seen as l i g h t  objective.  The t r i c h o c y s t s c o u l d be  s p i n d l e shaped elements on a dark  background.  A n a l y s i s o f Phenotypic Age Dependence C e l l s were grown i n the presence of excess food f o r f i v e days and were then l e f t a t room temperature u n t i l they entered  '  i  28 autogamy.  Twenty-one c e l l s were i s o l a t e d , f e d and allowed  t o complete one d i v i s i o n . was t e s t e d f o r autogamy.  One s i s t e r c e l l from each i s o l a t e The l i v i n g s i s t e r c e l l was c l o n e d .  Each day a s m a l l , newly d i v i d e d c e l l was t r a n s f e r r e d t o a f r e s h d e p r e s s i o n s l i d e and the number o f d i v i s i o n s completed by the c e l l s i n the o l d d e p r e s s i o n slidevwere r e c o r d e d . Every t h r e e days, a f t e r t h e t r a n s f e r of a young c e l l , the t h r e e p r e v i o u s d e p r e s s i o n s l i d e c l o n e s were pooled and t r a n s ferred to a p e t r i dish culture.  On the f o l l o w i n g day 100  d i v i d e r s were s e l e c t e d and d r i e d i n rows on s l i d e s . procedure was f o l l o w e d  This  so t h a t at approximately every 10  f i s s i o n s a sample of 100 d i v i d e r s f o r each l i n e was taken u n t i l t h e l i n e s were 60-70 f i s s i o n s o l d or u n t i l they went i n t o autogamy.  Test  f o r Mating R e a c t i v i t y C e l l s were grown i n tube c u l t u r e s w i t h excess f o o d f o r  s e v e r a l days.  A f t e r f i v e days the c u l t u r e s were allowed t o  exhaust the medium. t r a t e d by m i l d  c e n t r i f u g a t i o n and the c e l l p e l l e t was  i n a drop of D r y l ' s slide.  A f t e r 30-36 hours the c e l l s were concenplaced  s o l u t i o n i n the w e l l of a d e p r e s s i o n  C e l l s t h a t were mating r e a c t i v e would clump i n the  bottom of the w e l l .  Non-mating r e a c t i v e cells "would 1  disperse.  29 S y n c h r o n i z i n g Mating P a i r s Mating r e a c t i v e c u l t u r e s o f opposite mating type were mixed and l o o s e mating p a i r s were allowed t o form. mating c u l t u r e was l e f t of  The  f o r two hours a f t e r t h e f o r m a t i o n  the f i r s t l a s t i n g c e l l c o n t a c t s .  Couplets were then  drawn i n t o a l a r g e bore m i c r o p i p e t t e and e x p e l l e d  forcefully.  I f t h e p a i r remained t i g h t i t was c o n s i d e r e d t o be two hours old  (Berger  1969).  A l l p a i r s t h a t came, apart or appeared  l o o s e a f t e r e x p u l s i o n from t h e p i p e t t e were d i s c a r d e d . The s t a r t o f c o n j u g a t i o n was taken as t h e p o i n t a t which  cells  established l a s t i n g contact.  I n d u c t i o n of Macronuclear Regeneration Synchronous at  34.5°C  mating p a i r s were s h i f t e d t o a water bath  a t 5-g- h o u r s a f t e r  t h e s t a r t of c o n j u g a t i o n .  The p a i r s were kept a t the h i g h temperature f o r f i v e and were then r e t u r n e d t o room temperature.  hours  T h i s procedure  r e s u l t s w i n macronuclear r e g e n e r a t i o n i n a s m a l l percentage of  t h e exconjugants.  S e l e c t i o n of C e l l s Undergoing Macronuclear Regeneration (M.R.) Exconjugant  c e l l l i n e s undergoing M.R. were i d e n t i f i e d  by t h e use of s u i t a b l e g e n e t i c markers i n the p a r e n t a l macronuclei. (2)  The marker, pwA (Rung, 1971;  Chang et a l . ,  1974)  T h i s i s the time when the d i f f e r e n t i a t i n g synkaryon products a r e most v u l n e r a b l e t o shock treatment.  .30 was used i n one p a r e n t a l macronucleus and a mutation f o r t r i c h o c y s t nondischarge (nd6, the  other.  Homozygous pwA  tam A or tam G)was used i n  c e l l s f a i l t o show the t y p i c a l  a v o i d i n g r e a c t i o n of w i l d - t y p e c e l l s . l i n e s undergoing M.R.  Exautogamont  cell  were i d e n t i f i e d i n a d i f f e r e n t manner.  Exautogamonts were i s o l a t e d -into d e p r e s s i o n s l i d e and allowed t o complete two f i s s i o n s .  Two  cultures  of the f o u r  daughter c e l l s were d r i e d on s l i d e s , s t a i n e d and scored for  the presence of r e g e n e r a t i n g macronuclear fragments.  The Problem of Phenomic Lag When the f i l i a l  1  and p a r e n t a l genotypes d i f f e r , a c e r t a i n  amount of time i s r e q u i r e d b e f o r e the phenotype c o r r e s p o n d i n g to  the f i l i a l  genotype  comes i n t o e x p r e s s i o n .  T h i s i s be-  cause exconjugants r e t a i n the cytoplasm of t h e i r p a r e n t s . T h i s p e r s i s t e n c e of the p a r e n t a l phenotype  i n t o the F-^ gen-  e r a t i o n i s known as phenotypic or phenomic l a g (Sonneborn, 1953;  Berger, 1976)  and i s analogous t o the occurrence of  maternal e f f e c t s i n h i g h e r organisms.  Thus, when homozygous  tam A c e l l s are c r o s s e d t o w i l d - t y p e c e l l s ,  some of the  descendants of the tam A/tam A p a r e n t s w i l l show macronuclear m i s s e g r e g a t i o n at the f i r s t jugant.  or second d i v i s i o n of the excon-  M i s s e g r e g a t i o n i n tam,gives r i s e t o c e l l l i n e s which  have no macronuclei and which consequently undergo macronuclear regeneration.  The e x p r e s s i o n • o f the p a r e n t a l pheno-  31 type i n these M.R. of phenotypic  l i n e s can l e a d t o e r r o r s i n d e t e r m i n a t i o n  ratios.  To overcome the problem  of phenomic l a g , s i s t e r  produced at each of the f i r s t f i v e f i s s i o n s of the jugants were i s o l a t e d and t h e i r phenotypes (Figure 6 ) .  cells  excon-  were determined  L i n e s showing the p a r e n t a l (tarn) phenotype  were e l i m i n a t e d .  Cytological 1.  2.  Techniques  Fixative: a)  absolute ethanol  3 parts  b)  glacial acetic acid f i x f o r 20 min.  1 part  Whole c e l l mounts: A t h i n l a y e r of Meyer's egg albumin was  c l e a n microscope slide.  spread on a  s l i d e w i t h the b e v e l l e d edge of a second  The s l i d e was  heated g e n t l y over a bunsen burner  u n t i l the albumin steamed but was  not scorched.  C e l l s were  then p l a c e d i n microdrops, a l i g n e d i n rows on the prepared s l i d e , and allowed t o dry f l a t were then f i x e d and  on the s l i d e . ' The  stained.  3.  S t a i n s and s t a i n i n g procedures:  a)  Azure A  slides  F i g u r e 6.  I s o l a t i o n scheme of the F-^ progeny i n a c r o s s i n v o l v i n g a tarn gene. ma, macronucleus; mi, m i c r o n u c l e i ; f g , fragments. I s o l a t e s showing the p a r e n t a l were d i s c a r d e d .  (tarn) phenotype  34 10 mis of 0.5% Azure A  1.5 mis of 1 N 0.15  gms  HC1  of Sodium M e t a b i s u l p h i t e  Procedure: i) ii) iii) iv) v) vi) vii) viii) ix)  b)  Dippell  dry c e l l s on albuminized s l i d e s f i x f o r 20  h y d r o l y s e f o r 11 min. i n 1 N HC1 s t a i n f o r 15  counter  min.  stain  r i n s e i n E^O  (distilled)  a i r dry mount  stain p a r t s of acetoamine w i t h 4 . 5  Combine t h i s w i t h 2 p a r t s I'M  1% f a s t green i n 95% e t h a n o l .  c)  at 60°G  U^O  rinse with  Combine 10.5 Acetic acid.  min.  Feulgen Leucobasic f u s h s i n ( S c h i f f ' s Reagent) 100  mis  H0  0.5  gms  potassium m e t a b i s u l p h i t e  0.5  gms  basic  10  mis  1. N  2  (distilled)  fuchsin  HC1  HC1  p a r t s of 45% and 1 p a r t  35, B r i n g the water t o a b o i l and then c o o l t o 70°C.  Add dye  t o the water and a l l o w the s o l u t i o n t o c o o l t o room temperature.  Let mixture stand f o r s e v e r a l  brown b o t t l e .  hours i n a stoppered,  Add one gram d e c o l o u r i s i n g  s o l u t i o n f o r 1/2 hour.  c h a r c o a l and leave  Remove the c h a r c o a l by f i l t r a t i o n .  Store the s t a i n a t 4 C i n a brown b o t t l e  (light  sensitive  stain).  Acid bisulphite 0.5 gms  wash  potassium m e t a b i s u l p h i t e  5.0 mis  II N HC1  95.0 mis  H0 5  (distilled)  Procedure: i) ii)  dry  c e l l s on albuminized  f i x f o r 20 min. (ethanol:  slides g l a c i a l acetic  3:1) iii) iv) v) vi) vii) viii) ix) x) xi)  Hydrolyse f o r 11 min. a t 60°C i n 31 N HC1 r i n s e i n HgO stain f o r  (distilled)  30-60  rinse i n H 0 2  min.  ( d i s t i l l e d ) f o r 10 min.  r i n s e i n a c i d b i s u l p h i t e wash f o r 1 min. wash i n running water f o r 10 min. counter s t a i n a i r dry mount  acid  36 4.  Dryl's solution: S o l u t i o n A:  make stock t o 0.1 M  5.88 gms  sodium  2.76 gms  Na H" P0^  2.84 gms  Na  S o l u t i o n B:  make 0.1 M s t o c k .  2.22 gms  CaCl  2  H PO^  2  2  To make a 1 l i t r e  solution:  20 mis  sodium  10 mis  Na H  10 mis  Na  15 mis  CaCl  Mix t h e f i r s t  citrate  2  2  citrate  P0^  H P0^ 2  t h r e e s o l u t i o n s , add the d i s t i l l e d water t o  j u s t below the 1 l i t r e mark, add t h e C a C l t i l l e d water t o the 1 l i t r e mark.  /  2  and then:"add dis-  Ph t o 6.8.  37  RESULTS  I.  Mutagenesis The chemical mutagen n i t r o s o g u a n i d i n e  (N-methyl-  N ' - n i t r o - N - N i t r o s o g u a n i d i n e ) was used at a c o n c e n t r a t i o n of 75ug/ml f o r a p e r i o d of 60 minutes.  Seven separate mutagen-  e s i s experiments were run and a t o t a l of 12 v a r i a n t s were recovered (Table I I ) . A r e l a t i o n s h i p appears t o e x i s t  be-  tween the frequency of exautogamous death and the y i e l d of mutants (Table I ) . f a l l s below 45$,  As the frequency of exautogamous death  the y i e l d approaches zero; and as the f r e -  quency of exautogamous death i n c r e a s e s , t h e r e i s an i n c r e a s e i n the y i e l d of v a r i a n t s .  The sudden i n c r e a s e i n the f r e -  quency of exautogamous death i n experiments 6 and 7 (Table I) i  was a t t r i b u t e d t o the use of a new p r e p a r a t i o n of n i t r o s o guanidine t h a t was ation.  e v i d e n t l y more potent than the o l d p r e p a r -  The frequency w i t h which c e l l s underwent macronuclear  r e g e n e r a t i o n at the time of s e l e c t i o n was p r i m a r i l y caused by the c y t o t oxic e f f e c t of NTG and d i d not r e f l e c t of v a r i a n t l i n e s .  Presumably,  differences  the t o x i c e f f e c t of the  mutagen caused abnormal development  of the anlagen f o l l o w i n g  autogamy, and, as a consequence, macronuclear r e g e n e r a t i o n occurred.  38  i The s e l e c t i o n system used t o r e c o v e r these l i n e s the  same i n each experiment.  was  T h i s system was based on the  f a c t t h a t c e l l s undergoing macronuclear r e g e n e r a t i o n have a shortened c e l l c y c l e l e n g t h at the f i r s t and cycles following nuclear reorganization. the  second,cell  In w i l d type c e l l s ,  f i r s t c e l l cycle following nuclear reorganization i s  extended from f i v e hours (the normal v e g e t a t i v e c e l l  cycle  l e n g t h ) t o 11 t o 12 hours i n l e n g t h .  cycle  The second c e l l  i s a l s o extended t o S-g-—9 hours (Berger, 1 9 7 3 b ) .  This increase  in. the l e n g t h of the c e l l c y c l e could be a t t r i b u t e d t o the time r e q u i r e d f o r the new macronuclear anlagen t o r e a c h the DNA  content of a mature macronucleus.  C e l l s i n which the  p r e z y g o t i c macronuclear fragments have been r e l e a s e d from the  s u p p r e s s i v e i n f l u e n c e o f anlagen accumulate DNA  at a  g r e a t e r r a t e than do c e l l s undergoing normal n u c l e a r r e o r g a n ization  (Berger, 1 9 7 3 a ) .  That i s , the amount of DNA  synthes-  i z e d by the t o t a l complement of r e g e n e r a t i n g fragments exceeds the amount of DNA n u c l e a r anlagen. at  the minimum DNA  s y n t h e s i z e d by the growing macro-  T h e r e f o r e , r e g e n e r a t i n g c e l l s should a r r i v e content r e q u i r e d f o r c e l l d i v i s i o n b e f o r e  normally r e o r g a n i z i n g c e l l s .  Thus, the s e l e c t i o n system  i n v o l v e d the c o l l e c t i o n of e a r l y d i v i d e r s , a r i s i n g i n p e t r i d i s h c u l t u r e s , towards the end of the second c e l l  cycle.  D i v i d e r s c o u l d be found as e a r l y as 2 hours b e f o r e the expected end of the second c e l l p y c l e .  The r e c o v e r e d d i v i d e r s were  39 p l a c e d i n i s o l a t i o n w e l l s and allowed t o complete the c e l l division. the  One s i s t e r c e l l  from each i s o l a t e was cloned,  other was d r i e d on a microscope s l i d e , s t a i n e d , and  scored f o r the presence o f r e g e n e r a t i n g macronuclear fragment  For  the f i r s t  f o l l o w i n g the f i r s t  s i x experiments the s e l e c t i o n was done autogamy a f t e r mutagen treatment.  In  experiment 7, p a r t of the s e l e c t i o n was done f o l l o w i n g the f i r s t autogamy and part o f i t was done f o l l o w i n g the second autogamy a f t e r mutagen treatment. a f t e r the f i r s t  The v a r i a n t s r e c o v e r e d  autogamy were those which come i n t o phenotypi  e x p r e s s i o n p r i o r t o the f i r s t  f i s s i o n of the exautogamont.  T h i s occurs because phenomic l a g can be s u b s t a n t i a l l y reduced by the s t a r v a t i o n o f exautogamonts.(Sonneborn, Berger, 1976).  1953;  The mutagen-treated autogamous c u l t u r e s were  allowed t o s t a r v e f o r a few days b e f o r e  -Jre-f  any mutant geness t o come i n t o e x p r e s s i o n .  eeding t o a l l o w In experiment 7,  where the s e l e c t i o n was done f o l l o w i n g the second autogamy, t h e r e was no change of genotype and thus no phenomic l a g . It was hoped t h a t s e l e c t i o n a t the second autogamy would r e v e a l a wider range of v a r i a n t phenotypes than would be obtained from s e l e c t i o n a f t e r the f i r s t  autogamy a l o n e .  40 TABLE I MUTAGENESIS EXPERIMENTS: A COMPARISON OF THE FREQUENCY OF EXAUTOGAMOUS DEATH WITH THE YIELD  % EXAUTEXPERIMENT % VEGETA- OGAMOUS No. CELLS YIELD NUMBER TIVE DEATH DEATH (1) SCREENED (2) %  M.R.  AUTOGAMY  Ov-  1  20  40  1,500  1  3  ist  2  20  50  1,500  3  8  1st  3  30.  —  1,000  0  6  1st  4  20  45  1,000  0  18  1st  5  10  30  1,000  0  25  1st  - .6  99  99  100  4  40  1st  7  40  63  2,000  4  4  9  1st & 2n  (1)  The percent o f exautogamous death f o l l o w i n g the mutagenesis i s an i n d i c a t i o n of t h e ^ f r e q u e n c y o f l e t h a l mutations.  (2)  The y i e l d i s the number o f v a r i a n t  (3)  T y p i c a l l y , s e l e c t i o n was done f o l l o w i n g the f i r s t autogamy a f t e r the mutagenesis. In experiment 7 s e l e c t i o n was c a r r i e d out a f t e r the f i r s t and second autogamy f o l l o w i n g mutagenesis.  c e l l l i n e s recovered.  41  TABLE I I VARIANT LINES RECOVERED EXPERIMENT NUMBER  1  LINE  GENE  PHENOTYPE  46-6c  tam A  - M i s s e g r e g a t i o n o f anlagen a t nuclear reorganization - Abnormal d i s t r i b u t i o n of micronuclei at c e l l d i v i s i o n - Unequal d i v i s i o n , and miss e g r e g a t i o n of the macronucleus at c e l l d i v i s i o n - T r i c h o c y s t s misshaped and do not discharge  2  10-la  -  - Abnormal d i s t r i b u t i o n of micronuclei at c e l l d i v i s i o n  2  19-2b  -  - Abnormal d i s t r i b u t i o n of micronuclei at c e l l d i v i s i o n  6  21- 3(0 22- 5cJ 23- 2cO  - Abnormal n u c l e a r events a t conjugation  22-lc)  - Source o f amicronucleate  7  16- 2c) 17- l b )  7  40-4b  1  - Source of macronuclear r e generating c e l l s cells  - Slow growth  tam G  - S i m i l a r phenotype t o tam A - E x t r a n u c l e i found f o l l o w i n g nuclear reorganization  7  40-4b  2  sp_  - Production of large, black c e l l i n c l u s i o n s i n the a n t e r i o r p a r t of the c e l l .  42 I I . Genetic A n a l y s i s of the V a r i a n t s '  The  g e n e t i c b a s i s of the tarn A, tam. G and  sp_ (spot)  phenotypes were analysed by o u t - c r o s s i n g t o g e n e t i c a l l y marked w i l d type c e l l s .  The  F-^ phenotype was  with r e s p e c t t o a l l v a r i a n t t r i a t s  w i l d type  (tarn, sp. pw and  The l o s s of the pawn phenotype i n the  nd).  indicated that  true conjugation with c r o s s - f e r t i l i z a t i o n - h a d occurred. Fg g e n e r a t i o n was III).  The  obtained from F^ c e l l s by autogamy (Table  In both tarn.A and tam G c r o s s e s the t h r e e  macronuclear m i s s e g r e g a t i o n , non-discharge and pawn, a l l segregated w i t h a 1:1 complete concurrence  ratio.  traits,  of t r i c h o c y s t s There  was  of macronuclear m i s s e g r e g a t i o n  t r i c h o c y s t non-discharge  ( i n both tam A and tam G)  and suggesting  that the g e n e t i c b a s i s of both t r a i t s i s the same, s i n g l e , r e c e s s i v e gene. 1:1  S i m i l a r l y , sp and p_w segregated w i t h a  r a t i o from t h e i r w i l d type  Genetic a n a l y s i s was and 19-2b.  alleles.  not c a r r i e d out i n l i n e s  Both of these l i n e s were a c c i d e n t a l l y  when the c e l l s were f e d t o x i c c u l t u r e medium. a n a l y s i s of l i n e 21-3c  was  attempted, but was  10-la killed  Genetic not s u c c e s s f u l  owing t o the i n a b i l i t y of t h i s v a r i a n t t o complete c o n j u g a t i o n successfully. 3c  The r e s u l t s of one attempt t o o u t c r o s s  21-  t o g e n e t i c a l l y marked pw A c e l l s are g i v e n i n t a b l e IV.  From a t o t a l of 100  c o n j u g a t i n g p a i r s not one  s u c c e s s f u l l y completed  cross-fertilization.  conjugant  TABLE I I I A. Exautogamous  s e g r e g a t i o n of tam A heterozygote, tam A/tam A ;pw A/pw A  B. Exautogamous  s e g r e g a t i o n of tam G;sp double heterozygote, tam G/tam G ;pw A/pw A ;sp/sp'  C. Exautogamous  s e g r e g a t i o n of tam A;am double heterozygote, tam A/tam A ;pw A/pw A ;am/am'  LINE  A  1  46-6 c B  40-4b  6 46-6 c d4-43  +  +  2  1  20  23  tam G tam G tam G sp+ spjsp. pw A pw A+ pw A 11  12  9  tam A •"tarn A tam A am+ am am+ pwA pw A+ pw A  9 1. 2. 3. a. e.  12  2  -  13  +  tam A pw A+  x (3:l^  tam t a m  40.0  42  48  d  43  47  f  44  44  2  ....  51.1. 0 . 7 9 4  tam G tam G sp sp pw A+ pw A  +  13  tam G sp+ pw A+  +  14  12  tam A tam A am am pw A+ pw A 10  2  +  2$  11  +  REGOMB x ( l : l ) INANTS fo  >HEN0TY]ES  tam A tam A tam A pw A pw A+ pw A 22  +  +  3  F  +  f  tam A am+ pw A+  14  tam G sp_ pw A+  +  11 +  tam A am pw A+  8  tam G sp+ pw A  b  +  10 +  a  53.3  21.6l  c  52.3  27.28  e  0.O54  tam -A-- am+ pw A  9  0.375  x calculated using Yates' correction • No s e p a r a t i o n between m a c r o n u c l e a r m i s s e g r e g a t i o n and t r i c h o c y s t n o n d i s c h a r g e t r a i t s was observed Expectation f o r a recessive a l l e l e at a single locus E x p e c t a t i o n f o r a double mutant P*=.90. b.Pe^less t h a n . 0 5 , c . R ^ l e s s than . 0 5 , d . E * g r e a t e r t h a n . 7 0 Pocless t h a n . 0 5 , f . Ex g r e a t e r t h a n . 3 0  +  TABLE IV RESULTS OF MATING IN THE VARIANT 2 1 - 3 c  RESULTS  NUMBER OF PAIRS  Both conjugants dead  36  Cytogamy i n both conjugants  25 (25)  Conjugants f a i l e d t o mate  23 (23)  21-3c  13  exconjugant  pw A exconjugant  dead dead  Cross-fertilization TOTAL  (36)*  (13)  3 ( 3) 0 (  -)  100  () frequency as a percentage  J  1  45 Test f o r A l l e l i s m The newly obtained tam mutants, tam A and tam G. were c r o s s e d t o each other and t o other tam and t a m - l i k e mutants t o t e s t f o r a l l e l i s m .  In a l l cases t h e  g e n e r a t i o n was w i l d - t y p e f o r a l l t r a i t s [(table V ) . mutation,  The  pw A , was used as a marker i n the tam A and  tam G p a r e n t a l m a c r o n u c l e i . i n d i c a t e d that  Behaviour  o f t h e pw A marker  r e c i p r o c a l f e r t i l i z a t i o n occurred i n  all  cases.  Tam  A/tam A;jyti/am double mutant phenotype To determine  whether t h e e f f e c t s o f the mutations  were a d d i t i v e , tam A/tam A;am/am double homozygotes were r e c o v e r e d from the Fg progeny o f a mating between tam A and am homozygotes.  Approximately  25$ o f t h e F^  progeny showed very slow growth, ( the mean g e n e r a t i o n time was 2 0 . 0 0 - 0 . 4 5 [ S . E . ] hours, compared w i t h 7 . 3 7 0.20  [ S . E . ] hours f o r tam A/tam A and 6 . 2 5 - 0 . 1 6  hours f o r am/am). macronuclear  The slow growers d i s p l a y e d  m i s s e g r e g a t i o n and t r i c h o c y s t  both  non-discharge  t r a i t s and were assumed t o be the double mutant, VI).  [S.E.]  The frequency o f complete macronuclear  (table  missegregation  i n the presumed double mutants d i d not d i f f e r from the frequency o f m i s s e g r e g a t i o n observed f o r e i t h e r o f the mutant genes a l o n e .  i n c e l l s homozygous There was no e f f e c t  of c l o n a l age on the frequencyof macronuclear i n the double mutant.  missegregation  46  TABLE V COMPLEMENTATION TEST BETWEEN TAM GENES  GENE  TAM A  TAM G  +  -  tam 33  -  -  tam 8  -  -  -  -  -  +  tam A tam 38  tsm 21 f t A3 st A st B am:nd6 tam G  '  TABLE VI FREQUENCY OF COMPLETE MACRONUCLEAR MISSEGREGATION IN tam A/tam A;am/am DOUBLE MUTANT SAMPLE AGE NUMBER (number of fissions)  •gam A/tam A; am/ am  tam A/tam A  am/ am  10/30(33)  2/30(7)  20  1  7/30(23)*  30  2  5/30(17)  50  3  TOTAL  6/30(20)  5/30(17)  11/30(37)  . 14/30(47)  9/30(30)  23/90(26)  30/90(33)  16/90(18]  () frequency as a percentage  + A  -  48 I I I . Phenotypic A n a l y s i s A. Mutations a l t e r i n g the P a t t e r n o f Macronuclear  Division  1) E f f e c t s on V e g e t a t i v e C e l l s a) Generation time: The mean g e n e r a t i o n time of homozygous tam A c e l l s was 7.37±0.20[S.E.] hours as compared w i t h 4.80±0.09[S.E.] hours f o r w i l d t y p e . was s l i g h t l y  The mean g e n e r a t i o n time f o r tam G  s h o r t e r than f o r tam A, 6.29-0.27[S.E.]  hours  as compared w i t h 5.00^0.11[S.E.] hours f o r the w i l d type control.  The mean g e n e r a t i o n time f o r tam G i s very  c l o s e t o t h a t c a l c u l a t e d f o r am, 6 . 2 5 - 0 . l 6 [ S . E . ] hours. P r o b i t a n a l y s i s of the i n d i v i d u a l c e l l g e n e r a t i o n times of tam A r e v e a l s t h a t the c e l l c y c l e l e n g t h s a r e not normally d i s t r i b u t e d  (Figure 7 ) .  Approximately  of the c e l l s form a normally d i s t r i b u t e d  half  subpopulation  with a mean g e n e r a t i o n time of approximately 6.40 and have the same v a r i a n c e as w i l d type c o n t r o l s .  hours, The  upper p a r t o f the d i s t r i b u t i o n i s s t r o n g l y skewed t o the r i g h t , many c e l l s having very l o n g g e n e r a t i o n times, and some, presumably amacronucleate  c e l l s , do not d i v i d e  at a l l .  b) E f f e c t s on Shape and S i z e d i s t r i b u t i o n : A l a r g e percentage  of tam A c e l l s have an abnormal  FIGURE 7 .  P r o b i t a n a l y s i s of tam lengths. wild* type tam A  A cell  cycle  50  a3  N  Q3N  ' 51  f o o t b a l l shape compared t o the o v a l s l i p p e r shape o f w i l d type c e l l s  (figure 8 ) .  This difference  i s seen t o a l e s s e r extent i n tam G c e l l s .  i n shape There was  s u b s t a n t i a l v a r i a t i o n i n the s i z e o f both d i v i d i n g and morphostatic c e l l s i n c u l t u r e s and tam G c e l l s . analysis stained  o f both homozygous tam A  A b s o r p t i o n microspectrpphpibicametric  o f .Feulgen-stained and Napthol-Yellow c e l l s showed t h a t  p r o t e i n ) and macronuclear twice t h a t  both cytoplasmic mass  counter(total  DNA content were approximately  f o r wild-type c e l l s .  The mean p o s t r - f i s s i o n  cumulative e x t i n c t i o n f o r macronuclear f o r tam A/tam A c e l l s ; 100.7-5.87  f  o  r  DNA was 206.6i9.14  wild-type  cells  and 205.0-22.75 f o r the double mutant tam A/tam A; am/am. The cumulative e x t i n c t i o n o f t o t a l p r o t e i n was 165.0^ 14 f o r tam A/tam A and 90- 16 f o r w i l d - t y p e c e l l s .  c) E f f e c t s on macronuclei i ) Phenotype The  sequence o f morphogenetic changes which the  macronuclei o f both tam A/tam A and tam G/tam G undergo d u r i n g f i s s i o n was determined  from a s e r i e s o f s t a i n e d  c e l l s f i x e d a t v a r i o u s stages o f f i s s i o n .  The f o l l o w i n g  analysis  affected  d e a l s o n l y w i t h the most s e v e r e l y  found a t each stage o f d i v i s i o n ( f i g u r e 9 ) .  nuclei  Expression  of both the tam A and tam G phenotypes i s v a r i a b l e and  FIGURE 8. A comparison of c e l l  morphology.  53  FIGURE 8  F i g u r e 9 . M i s s e g r e g a t i o n of the macronucleus a t f i s s i o n i n c e l l s e x p r e s s i n g the tam  phenotype.  Ma, macronucleus; mi, micronucleus;  f v , food  vacuoles A. Normal d i v i s i o n of the macronucleus i n tam/ tam c e l l s not e x p r e s s i n g the tam B. Complete m i s s e g r e g a t i o n  phenotype.  of the macronucleus  at f i s s i o n i n c e l l s e x p r e s s i n g the tam phenotype.  FIGURE 9  56 almost h a l f the c e l l s i n p o p u l a t i o n s of tam A/tam A and tam G/tam G c u l t u r e s d i v i d e n o r m a l l y . the  By examining  most extreme phenotypes at each stage of c e l l  division  i t was hoped t h a t the sequence o f events l e a d i n g t o e i t h e r p a r t i a l or complete m i s s e g r e g a t i o n of the macronucleus c o u l d be e s t a b l i s h e d .  The m a c r o n u c l e i of homozygous tam A and tam G c e l l s migrate t o the c e n t r a l p a r t o f the c e l l at about age 0.7. controls.  interfission  T h i s corresponds t o o b s e r v a t i o n s on w i l d - t y p e However, u n l i k e w i l d - t y p e c e l l s , the macro-  n u c l e i of tam A/tam A and tam G/tam G c e l l s remain rounded and do not continue w i t h the subsequent morphog e n e t i c events which occur i n w i l d type c e l l s plate 1).  ( f i g u r e 10,  T h i s l a c k of e l o n g a t i o n and proper placement  of the macronucleus of the tam mutants l e a d s t o v a r y i n g degrees of unequal d i v i s i o n of the macronucleus  (partial  m i s s e g r e g a t i o n , p l a t e 2) between s i s t e r c e l l s and, i n some cases, r e t e n t i o n o f the e n t i r e p r e f i s s i o n macronucleus by one of the s i s t e r c e l l s  ( complete macronuclear mis-  s e g r e g a t i o n , p l a t e 1, G).  Where g r o s s l y unequal d i v i s i o n  of  the macronucleus occurs, the l a r g e r p o r t i o n i s always  r e t a i n e d by the a n t e r i o r c e l l  ( p r o t e r ) , the p o s t e r i o r  ( o p i s t h e ) r e c e i v i n g the s m a l l e r p o r t i o n ( p l a t e  3,D).  cell  F i g u r e 1 0 . A comparison of macronuclear p r e d i v i s i o n morphogenesis between w i l d - t y p e and homozygous tam ma,  cells.  macronucleus; mi, micronucleus; f v , food  vacuoles The decimal f r a c t i o n s i n d i c a t e the approximate interfission  age.  58  ma  I  59 Complete m i s s e g r e g a t i o n of the macronucleus always occurs ( p l a t e 1,G; Macronuclear  t o the p r o t e r  p l a t e 2, A; p l a t e 3, A ) .  s l i p p a g e has been observed i n other tam  mutants, but was  not observed i n e i t h e r tam A or tam G  ( R u i z , et a l . , 1976).  In the most severe cases the macronuclei of homozygous tam A and tam G c e l l s do not d i v i d e at a l l , e i t h e r d u r i n g c y t o k i n e s i s or immediately t h e r e a f t e r .  Macronuclear  e l o n g a t i o n and c o n s t r i c t i o n i s e n t i r e l y absent. m i s s e g r e g a t i o n of such a macronucleus  r e s u l t s from the  p h y s i c a l t e a r i n g of the u n d i v i d e d macronucleus advancing f i s s i o n furrow.  Partial  by the  In c e l l s e x p r e s s i n g the  tam  phenotype t o a l e s s e r degree, s l i g h t e l o n g a t i o n of the macronucleus  i s e v i d e n t , even w h i l e the  macronucleus  remains i n a c e n t r a l p o s i t i o n w i t h i n the c e l l - w h i l e macronuclear  c o n s t r i c t i o n may  slightly elongated macronucleus of c y t o k i n e s i s .  or may  not occur.  rounds up a t the completion  M i s p l a c e d macronuclei may  c o n s t r i c t , but the c o n s t r i c t i o n may  elongate and  be at a d i f f e r e n t  l a t i t u d e than the l i n e of s e p a r a t i o n of the two cells  ( p l a t e 4,  A).  of s m a l l fragments of the s i s t e r c e l l s  The  daughter  T h i s can r e s u l t i n the f o r m a t i o n of the macronucleus ( p l a t e 4,  whether these macronuclear  B and C ) .  fragments  i n one or other I t i s not known  segregate at the  60 next c e l l d i v i s i o n and, a fully  developed  i f they do, whether each becomes  macronucleus.  The p a t t e r n of m i s s e g r e g a t i o n of the m a c r o n u c l e i of homozygous tam A and homozygous tam G c e l l s i s not the same.  Tam  G shows a h i g h e r frequency of p a r t i a l  m i s s e g r e g a t i o n and a lower frequency of complete  mis-  s e g r e g a t i o n when compared w i t h tam A (Table V I I ) .  i'i) Penetrance a) Age  and E x p r e s s i o n  Effects:  (tam A only)  Since the degree varied  of macronuclear  missegregation  c o n s i d e r a b l y between d i f f e r e n t experiments,  age dependence' of phenotypic e x p r e s s i o n was Age  examined.  dependent e x p r e s s i o n had been demonstrated  f o r a s i m i l a r mutant, am  ( N o b i l i , 1959  and  A s e r i e s of c e l l l i n e s were s t a r t e d  the  previously  1961).  from exautogamonts.  At approximately 10 f i s s i o n i n t e r v a l s samples of  100  d i v i d e r s were r e c o v e r e d and s t a i n e d , and the frequency of macronuclear and 12). remained  m i s s e g r e g a t i o n was  determined  ( F i g u r e s 11  The o v e r a l l p a t t e r n of macronuclear almost  macronuclear  constant.  missegregation  However, examination  of  complete  m i s s e g r e g a t i o n alone r e v e a l e d a change i n  the frequency w i t h c l o n a l age.  Between 10 and 30 f i s s i o n s  61  TABLE VII A COMPARISON OF THE FREQUENCY OF PARTIAL AND COMPLETE MACRONUCLEAR MISSEGREGATION IN tam A AND tam G  SAMPLE SAMPLE No. OF SIZE No. FISSIONS  PARTIAL tam A  tam G  COMPLETE tam G tam A  25  1  50  5/50 (t©?» )24/50(48) 17/50(34) 3/50.(6)  35  2  50  4/50(  8) 21/50^42) 18/50(36) 6/50(12)  () frequency as a percentage  F i g u r e 11.  Penetrance of the f u n c t i o n of c l o n a l A l l t r a i t s are p a r t i a l and  tam  and  am  genes as  a  age.  i n c l u d e d i n t h i s datum, i . e ;  complete macronuclear m i s s e g r e g a t i o n ,  abnormal d i s t r i b u t i o n of m i c r o n u c l e i . V e r t i c a l bars i n d i c a t e  95f°  confidence  intervals  63  F i g u r e 12. The frequency o f complete macronuclear miss e g r e g a t i o n i n tam' A/tam A and am/am c e l l s as a f u n c t i o n o f c l o n a l age. •-•  am/am (my data)  A-A  am/am ( N o b i l i , 1 9 6 1 )  V e r t i c a l bars i n d i c a t e 95% c o n f i d e n c e i n t e r v a l s  6$  66 the frequency of complete  m i s s e g r e g a t i o n remained q u i t e  low (approximately 2 0 $ ) .  At. 40 f i s s i o n s t h e r e was  dramatic i n c r e a s e i n the frequency of complete which dropped  a  missegregation  o f f only s l i g h t l y w i t h i n the next 20  The same experiment  was  fissions.  c a r r i e d out c o n c u r r e n t l y w i t h  homozygous am c e l l s f o r comparison.  The degree  of  e x p r e s s i o n of tam A i s g e n e r a l l y g r e a t e r than t h a t f o r am,  e s p e c i a l l y i n young c l o n e s .  Homozygous am  cells  showed a g r e a t e r age dependency than d i d homozygous tam A cells.  "  ^  b) Temperature e f f e c t s : Homozygous tam A c e l l s and homozygous tam G c e l l s were grown at e i t h e r 34.5°C or 27.0°C f o r 2 4 . 0 0 hours. D i v i d i n g c e l l s from both temperature  groups were c o l l e c t e d ,  d r i e d on s l i d e s , s t a i n e d , and scored f o r both m i c r o n u c l e a r and macronuclear  m i s s e g r e g a t i o n (Table V I I I ) .  In tam G  c e l l s , m i s s e g r e g a t i o n of the macronucleus d i d not at 34.5°C, while at the c o n t r o l temperature 50$ macronuclear e f f e c t was  missegrggation occurred.  l e s s apparent  occur  (27.0°C) The  i n homozygous tam A  temperature  cells.  M i s s e g r e g a t i o n of both types of n u c l e i occurred at 34»5°C and 2 7 . 0 ° C .  However, the frequency of abnormal d i s t r i b u t i o n s  of m i c r o n u c l e i and macronuclear higher at 27.0°C.  m i s s e g r e g a t i o n was  10%  67  TABLE V I I I THE EFFECT OF TEMPERATURE ON NUCLEAR MISSEGREGATION IN HOMOZYGOUS tam A AND tam G CELLS  GENOTYPE  tam A tam A tam G tam G  FREQUENCY OF MACRONUCLEAR MISSEGREGATION  27.0 C 34.5 C 39/100(391 29/100(29) 17/34(50)  0/34(0)  FREQUENCY OF MICRONUCLEAR MISSEGREGATION 2 7 . 0 C. 34.5  C 26/100(26) 15/100(15) 5/34(15)  () frequency as a percentage  0/34(0)  68 d) E f f e c t s on M i c r o n u c l e i The  numbers of m i c r o n u c l e i observed i n tam A/tam A  and tam G/tam G c e l l s v a r i e d c o n s i d e r a b l y when compared t o the number i n w i l d type c e l l s c l e a r l y shown i n amicronucleate  (Table I X ) . T h i s i s c e l l s where the micro-  n u c l e i are not obscured i n the r e g i o n of the macronucleus (Plate 5).  The d i s t r i b u t i o n of m i c r o n u c l e i a t d i v i s i o n  i n homozygous tam c e l l s was determined by a n a l y s i s of the numbers of m i c r o n u c l e i i n r e l a t e d p a i r s of s i s t e r ( F i g u r e 13).  cells  The number of m i c r o n u c l e i was compared  between s i s t e r d i v i d e r s and the p a r e n t a l complement was a c s e r t a i n e d  (Table X ) .  c o n s i s t e n t with the hypothesis  micronuclear-  The r e s u l t s a r e  t h a t abnormal numbers of  m i c r o n u c l e i a r i s e through improper d i s t r i b u t i o n of the micronuclei at c e l l d i v i s i o n  (Figure 14).  This  hypothesis  was d i r e c t l y confirmed by a n a l y s i s of the d i s t r i b u t i o n of m i c r o n u c l e i  i n stained, d i v i d i n g c e l l s .  The d i v i d i n g  c e l l s were c l a s s i f i e d e i t h e r as e a r l y , as middle or as l a t e d i v i d e r s and the p o s i t i o n of the n u c l e i i n each was noted (Figure  cell  15).  Proper e l o n g a t i o n of micronuclear does not appear t o occur  division  spindles  i n tam A/tam A c e l l s and the  m i c r o n u c l e i remain c l u s t e r e d i n the c e n t r a l r e g i o n of the cell  ( F i g u r e 16, P l a t e 3, A ) .  Whereas i n w i l d type  cells,  69  TABLE IX COMPARISON OF THE NUMBER OF MICRONUCLEI BETWEEN HOMOZYGOUS tam A AND tam G CELLS AND WILD-,TYPE GENOTYPE  NUMBER  2 +  A  2  96/100(96)  1  OF  MICRONUCLISI  ^2  >2  obscured  -  -  4/100(4)  tam A^ tam A  30/75(40)  4/75(5)  tam G^ -tam G  45/60(75)  0/60(0) ' 11/60(18)  29/75(39)  1) () frequency as a percentage 2) approximately 40 f i s s i o n s o l d 3) between 25-35 f i s s i o n s o l d  12/75(16) 4/60(7)  I  Figure 13.  Diagram of the  experiment comparing  number of m i c r o n u c l e i sister A.  the  in related pairs  of  cells.  Isolated  d i v i d e r allowed t o complete d i v i s i o n  B. S i s t e r c e l l s separated and the next c e l l c y c l e  (C and  D. P a i r s of r e l a t e d s i s t e r  allowed t o D)  c e l l s dried i n  microdrops on albuminized s l i d e s  complete  71  FIGURE 13  72 TABLE X MICRONUCLEAR SEGREGATION AT CELL DIVISION.  A COMPARISON  BETWEEN RELATED SISTER DIVIDERS IN tam A/tam A , tam G/tam G AND WILD^TYPE CELLS MICRONUCLEAR MICRONUCLEAR NUMBER OF COMPLEMENT DISTRIBUTION ffam A OF PARENT BETWEEN tam A DIVIDER RELATED SISTERS (reconstructed) ** 2-2 2-2; 2-2 35/65(54)  SISTER DIVIDERS tam G + tam G +  78/120(70) 97/100  -  -  2-2  1-3, 2-2  4/65(6)*  2-2 .  1-3; 1-3  4/65(6)  2-2  0-4; 2-2  1/65(2)  2-2  0-4* 1-3  -  1/120(1)  -  2-2  0-4; 0-4  2/65(3)  9/120(8)  -  3-3  3-3;  6/65(9})  12/120(10)  -  3-3  3-3; 2-4  1/120(1)  -  3-3.  ** 3-3., 1 ^ 5  1/65(2)  1-3  3-3 1-1  1/65(2)  2/120(2)  -  1-3  2-0 3-3  -  1/120(1)  -  4-4  4-4, 4-4  1/65(2)  3/120(3)  -  5-5  1/65(2)  3/120(3)  -  6-6  5-5 5-5 6-6; 6-6**  1-1  1-1 •1-1  5/65 (8)  1-1  1-1 >2-0  **  3-3  -  ** **•  1/65(2) J  **  2/65(3)  2/120(2)  -  -  8/120(7)  -  J>() frequency as a percentage *Plate 6  -  -  -  Figure 14.  T h e o r e t i c a l diagram demonstrating how numbers of m i c r o n u c e l i may e x p r e s s i n g t h e tam ma,  large  arise i n cells  phenotype.  macronucleus; mi, m i c r o n u c l e i ; f v , food vacuoles  F i g u r e 1$. A. A comparison of e a r l y , middle and l a t e  dividers.  B. D i v i s i o n of a d i v i d i n g c e l l i n t o f i e l d s f o r the comparison of m i c r o n u c l e a r between tam/tam and w i l d - t y p e af, anterior cf,  field;  amf,  c e n t r a l f i e l d ; pmf,  pf, p o s t e r i o r  field  localization, dividers.  anterior  mid-field  posterior  mid-field  FIGURE 15  F i g u r e 16. I l l u s t r a t i o n  of the d i f f e r e n c e  i n micronuclear  placement i n w i l d - t y p e and tam A/tam A d i v i d i n g cells. ma,  macronucleus; mi, micronucleus;  f v , food  vacuoles The decimal f r a c t i o n s i n d i c a t e the approximate interfission  j  age  tam tam  FIGURE 16  79 the s p i n d l e s elongate and the m i c r o n u c l e i migrate towards the p o l e s of the c e l l .  T h i s i s not t r u e f o r a l l homozygous  tam A and tam G c e l l s , but only f o r those t h a t a r e e x p r e s s i n g the tam phenotype.  There was a s l i g h t temperature  e f f e c t on the s e g r e g a t i o n  of m i c r o n u c l e i , a t c e l l d i v i s i o n , i n tam A/tam A c e l l s . The frequency o f m i s s e g r e g a t i o n was i n c r e a s e d by 10% a t 27.0°C. as compared t o the observed frequency at j54.5°C. In homozygous tam G c e l l s the temperature s l i g h t l y more pronounced.  effect  was  No m i c r o n u c l e a r m i s s e g r e g a t i o n  occurred at 34«5°C. as compared w i t h a frequency o f  14.5%  at 27.0°C.  e) E f f e c t  on T r i c h o c y s t s  The t r i c h o c y s t s of both homozygous tam A and tam G c e l l s do not d i s c h a r g e when the c e l l s are k i l l e d w i t h picric  acid.  W i l d type c e l l s always d i s c h a r g e t h e i r  t r i c h o c y s t s under these c o n d i t i o n s .  Observations on  b u r s t c e l l s , w i t h n e g a t i v e phase c o n t r a s t o p t i c s , r e v e a l s t h a t abnormally shaped t r i c h o c y s t s are present i n the cytoplasms  o f tam A/tam A and tam G/tam G c e l l s  (Figure  In homozygous tam A c e l l s , the morphology of the t r i c h o c y s t s i s s i m i l a r t o t h a t of the f o o t b a l l - s h a p e d v a r i e t y d e s c r i b e d by P o l l a c k (1974).  L i k e the f o o t b a l l -  17).  Figure 17.  Illustration  showing the d i f f e r e n c e i n  t r i c h o c y s t morphology. A. Wild-type B. tam A/tam A C. tam  G/tam G  (The w i l d - t y p e t r i c h o c y s t s are almost twice the l e n g t h of the f o o t b a l l - s h a p e d and stubby trichocysts.) * i  normal  38,  ooo  football  32,000  stubby  FIGURE 17  82 shaped t r i c h o c y s t d e s c r i b e d  by P o l l a c k  (1974), the  abnormal t r i c h o c y s t s i n tam A/tam A c e l l s tend t o d i s s o l v e when the c e l l i s b u r s t .  In p r e p a r a t i o n s of burst  type c e l l s , the t r i c h o c y s t s do not d i s s o l v e . or c o r t i c a l l y  No  wildsurface  a t t a c h e d t r i c h o c y s t s a r e found i n tam A/tam A  c e l l s which i n d i c a t e s a f a i l u r e migrate w i t h i n the c e l l ' s  of the t r i c h o c y s t s t o  cytoplasm.  In homozygous tam G c e l l s the morphology of the t r i c h o c y s t s i s s i m i l a r t o the stubby v a r i e t y by P o l l a c k  (1974)•  described  Although no t r i c h o c y s t s are seen t o  discharge when c e l l s are k i l l e d w i t h p i c r i c  a c i d , a few  t r i c h o c y s t s may d i s c h a r g e when c e l l s are burst pressure. football  under  Stubby t r i c h o c y s t s do not d i s s o l v e , u n l i k e the t r i c h o c y s t s of tam A/tam A c e l l s .  2)  E f f e c t s During Nuclear R e o r g a n i z a t i o n  a)  Autogamy: Both i n tam A/tam A and tam G/tam G c e l l s the n u c l e a r  events d u r i n g autogamy are normal.  The m i c r o n u c l e i  undergo  m e i o s i s , a synkaryon i s formed from f u s i o n of the two p r o n u c l e i , and the synkaryon completes two p o s t z y g o t i c t b o g i v e r i s e t o the presumptive m i c r o n u c l e i anlagen.  divisions  and macronuclear  In w i l d type exautogamonts, s e g r e g a t i o n of the two  macronuclear anlagen, without d i v i s i o n ,  occurs a t the f i r s t  fission  83 f o l l o w i n g n u c l e a r r e o r g a n i z a t i o n , r e s t o r i n g the v e g e t a t i v e n u c l e a r complement of one macronucleus and two m i c r o n u c l e i per c e l l .  In tam A/tam A and tam G/tam G ' c e l l s , however,  both anlagen may segregate first  t o one daughter c e l l a t the  c e l l d i v i s i o n , l e a d i n g t o r e g e n e r a t i o n of the  macronuclear fragments i n t h e other daughter c e l l 18b, of  Plate 7,  C,D, and E ) .  both anlagen  Although  (Figure  In most cases where r e t e n t i o n  occurred i n one c e l l , i t was the p r o t e r .  missegregation  of anlagen  occurred a t the f i r s t  f i s s i o n f o l l o w i n g n u c l e a r r e o r g a n i z a t i o n , the occurrence was r a r e . at  More commonly, s e g r e g a t i o n of anlagen was normal  the f i r s t f i s s i o n , but m i s s e g r e g a t i o n  occurred with a h i g h frequency  of the macronucleus  at the second f i s s i o n .  ( F i g u r e 18 a ) .  Since the frequency missegregation  of complete macronuclear  d u r i n g v e g e t a t i v e c e l l d i v i s i o n was age  dependent, the p o s s i b i l i t y was c o n s i d e r e d t h a t the frequency age  of m i s s e g r e g a t i o n  dependency.  of anlagen may a l s o show an  That i s , the o l d e r the c e l l s are when  they e n t e r autogamy, the h i g h e r the frequency s e g r e g a t i o n of anlagen reorganization. of  of mis-?-  may be f o l l o w i n g n u c l e a r  To t e s t f o r t h i s p o s s i b i l i t y , c u l t u r e s  homozygous tam A c e l l s of d i f f e r e n t ages were s t a r v e d  and allowed t o enter autogamy.  Exautogamonts were i s o l a t e d  Figure 18. I l l u s t r a t i o n of the experiment the  comparing  frequency of macronuclear m i s s e g r e g a t i o n  at, the f i r s t A. Complete  and second exautogamous  division.  m i s s e g r e g a t i o n of the macronucleus  at the second exautogamous  division.  B. M i s s e g r e g a t i o n of one anlage a t the f i r s t ' exautogamous  division.  86 i n t o f r e s h medium and allowed t o d i v i d e t w i c e .  After  the  first  f i s s i o n , daughter c e l l s were s e p a r a t e d .  the  second f i s s i o n , two p a i r s of daughter c e l l s were d r i e d  on s l i d e s and s t a i n e d w i t h Azure A (Figure 1 8 ) .  After  The  frequency of m i s s e g r e g a t i o n a t both the f i r s t and second f i s s i o n a f t e r autogamy, was then determined  (Table X I ) .  Increased p a r e n t a l age does not l e a d t o an i n c r e a s e i n the frequency of m i s s e g r e g a t i o n of macronuclear anlagen a f t e r autogamy.  b) Conjugation: Homozygous tam A and tam G c e l l s form normal pairs.  mating  No unusual n u c l e a r events were observed i n  s t a i n e d c o n j u g a t i n g p a i r s of tam A/tam A c e l l s .  Mis-  s e g r e g a t i o n of macronuclear anlagen o c c u r r e d at both the  first  and second f i s s i o n f o l l o w i n g c o n j u g a t i o n .  Abnormal n u c l e a r events o c c u r r e d o c c a s i o n a M y j y i n tam G/ tam G c e l l s g i v i n g r i s e t o an excess number of m i c r o n u c l e i and/or macronuclear anlagen.  In cases where excess  numbers of n u c l e i were p r e s e n t , between 3 and 4 anlagen and 4 and 6 m i c r o n u c l e i were the most common complements. To determine what abnormal n u c l e a r events o c c u r r i n g a t c o n j u g a t i o n were r e s p o n s i b l e f o r the excess numbers of n u c l e i i n exconjugants, a number of c o n j u g a t i n g p a i r s were d r i e d on s l i d e s ,  stained,,and examined f o r unusual  87  TABLE XI THE FREQUENCY OF MISSEGREGATION OF ANLAGEN AT THE FIRST AND SECOND POST-AUTOGAMOUS CELL DIVISION  PARENTAL AGE (fissions)  No.OF CELL LINES  NUMBER OF DIVIDERS SHOWING MISSEGREGATION OF ANLAGEN first fission second f i s s i o n  35  50  15(30)*  21(42)  40  50  4( 8)  12(24)  50  40  8(20)  8(20)  60  . 40  4(10)  11(28)  70  25  5(20)  7(28)  () frequency as a percentage  88 nuclear patterns.  I t was thought t h a t e i t h e r the number  of excess n u c l e i r e s u l t e d from e x t r a synkaryon d i v i s i o n s , or e x t r a d i v i s i o n s of one or more of the synkaryon products, or t h a t , the number o f excess n u c l e i may r e s u l t from the p e r s i s t e n c e of some of the h a p l o i d m e i o t i c products, which n o r m a l l y degenerate.  Analysis of conjugating  p a i r s showed t h a t the proper number of h a p l o i d products  (7)  d i d not degenerate, but p e r s i s t e d through the stage o f d i f f e r e n t i a t i o n o f the synkaryon p r o d u c t s .  Among the c o n j u g a t i n g p a i r s d r i e d , and s t a i n e d f o r a n a l y s i s was one p a i r i n which an amacronucleate  cell  was coupled t o a normal conjugant ( P l a t e 3,C) i n a t y p i c a l mating c o n f i g u r a t i o n .  Four s m a l l n u c l e i were  observed i n the amacronucleate p a r t n e r .  Two of these  n u c l e i were s l i g h t l y s m a l l e r and more f a i n t l y These two appeared t o be completing a d i v i s i o n  stained. (second  synkaryon d i v i s i o n ? Or f i r s t hemikaryon d i v i s i o n ? ) . The remaining two n u c l e i were s l i g h t l y l a r g e r and more darkly staining  (one p l a c e d a n t e r i o r l y , the other  p o s t e r i o r l y p l a c e d , P l a t e 3,C). These two n u c l e i may be two o f the h a p l o i d products t h a t p e r s i s t e d through con- • j u g a t i o n or they may be d i v i s i o n products of the synkaryon. Reference t o amacronucleate conjugants has not been found i n the l i t e r a t u r e .  89 O c c a s i o n a l l y , i n homozygous tam A or homozygous tam G exconjugants, development of c e r t a i n products of the synkaryon was abnormal.  In some cases one or  both anlagen were u n d e r s i z e d and f a i l e d t o s u r p r e s s DNA s y n t h e s i s i n the macronuclear  fragments  r e g e n e r a t i o n i n evidence^ P l a t e 9,B). normal  (macronuclear In other cases,  s i z e d anlagen were found i n the presence of  e a r l y r e g e n e r a t i n g macronuclear  fragments  Exconjugant tam A or tam G c e l l s ,  ( P l a t e 9, Aand C ) .  undergoing  macronuclear r e g e n e r a t i o n , underwent normal r e g e n e r a t i o n . The fragments a l i g n e d a l o n g the d o r s a l c o r t e x , elongated d u r i n g c e l l d i v i s i o n and segregated a t c e l l  division,  approximately one h a l f the number migrated t o each cell  ( P l a t e 9,D).  In some cases, m i s s e g r e g a t i o n of the  r e g e n e r a t i n g fragments  occurred.  In such cases one s i s t e r  c e l l r e c e i v e d a l l or most of the fragments  ( u s u a l l y the  p r o t e r ) w h i l e the other r e c e i v e d no fragments one fragment  sister  ( P l a t e 9,E).  or o n l y  Regenerating fragments  usually  grow at approximately the same r a t e , so a l l the fragments w i t h i n one c e l l are approximately the same s i z e .  However, ,  t h i s was not always the case i n tamA/tam A c e l l s , where sometimes the r e g e n e r a t i n g fragments were of very, unequal size  ( P l a t e 9,F).  90 B. V a r i a n t s w i t h a l t e r e d M i c r o n u c l e a r D i s t r i b u t i o n L i n e s 1 0 - l a . 19-2b 1)  and  21-3c  E f f e c t s on V e g e t a t i v e c e l l s  a) Generation time; The mean g e n e r a t i o n time f o r l i n e 10-la was [ S . E . ] , f o r l i n e 19-2b l i n e 21-3c  was  6.12^.08  5.35±.14[S.E.] hours, f o r  the mean g e n e r a t i o n time was  hours compared t o 5 . 0 0 i . l l [ S . E . ]  19.20-«26[S.E.]  hours f o r w i l d t y p e .  The c e l l c y c l e l e n g t h i n l i n e s 10-la and 19-2b  was  extended  only s l i g h t l y from the normal w i l d type c e l l c y c l e l e n g t h , whereas the d e l a y i n the l e n g t h of the c e l l c y c l e f o r the v a r i a n t 21-3c  was c o n s i d e r a b l e .  b) E f f e c t s on the N u c l e i :  .  D i v i s i o n of the macronucleus was v a r i a n t s , w i t h each daughter  normal i n a l l t h r e e  c e l l receiving  h a l f of the p r e f i s s i o n macronucleus. macronuclear  v  ;l  approximately  P a r t i a l or  m i s s e g r e g a t i o n at c e l l d i v i s i o n was  observed i n any of these v a r i a n t s .  complete not  Observations made on  samples of s t a i n e d i n t e r f i s s i o n c e l l s r e v e a l e d a v a r i a b l e number of m i c r o n u c l e i , i n s t e a d of the normal w i l d type complement of two. h i g h (5,  6 and 7)  In l i n e s 10-la and 19-2b  abnormally  and abnormally low '§0 and.l) numbers of  m i c r o n u c l e i were found 3.6%  both  (Table X I I ) .  In l i n e 2 l - 3 c ,  of the c e l l s contained the normal m i c r o n u c l e a r  only  91  TABLE X I I VARIATION IN THE NUMBER OF MICRONUCLEI IN MORPHOSTATIC OF LINES 10-la AND 19-2b  LINE NUMBER  NUMBER  10-la  49/75(65)*  19-2b  30/80(38)  51-s  94/96(98)  OF  MICRONUCLEI  >2  12  8/75(11)  18/75(24)  11/80(13)  39/80(49)  2/96( 2)  -  () frequency as a percentage  CELLS  92 complement, 83.0$ had only a s i n g l e micronucleus 13.4$  and  had no m i c r o n u c l e i .  I t was c o n s i d e r e d t h a t the excess m i c r o n u c l e i i n l i n e s 10-la and 19-2b  found  might a r i s e through e i t h e r e x t r a  d i v i s i o n s of the m i c r o n u c l e i at the end of each c e l l or,  cycle,  t h a t the e x t r a numbers of m i c r o n u c l e i might a r i s e  through m i s s e g r e g a t i o n  of the m i c r o n u c l e i at c e l l  division.  A n a l y s i s of s t a i n e d , d i v i d i n g s i s t e r c e l l s allowed f o r the r e c o n s t r u c t i o n of the m i c r o n u c l e a r the  parent  dividers.  complement i n  The r e s u l t s were c o n s i s t e n t w i t h  the l a t t e r p o s s i b i l i t y ( T a b l e X I I I ) .  c)Penetrance  and E x p r e s s i o n :  i ) Age E f f e c t s ,  ( l i n e s 10-la and 19-2b)  T  Since phenotypic  e x p r e s s i o n i s o f t e n a l t e r e d by  p h y s i c a l or b i o c h e m i c a l f a c t o r s , the e f f e c t of age and temperature on e x p r e s s i o n i n l i n e s 10-la and 19r2b was examined.  Exautogamous i s o l a t e s were made and c l o n e d .  Samples of d i v i d i n g c e l l s were taken at 10 i n t e r v a l s and the m i c r o n u c l e a r ( F i g u r e 20). expression.  or o l d e r .  complement was  analysed  Line 10-la d i d not show age dependent In l i n e 19-2b,  micronuclear missegregation old  fission  however, the frequency of i n c r e a s e d i n c e l l s 50  There was an age dependent s h i f t  fissions  from  93 TABLE X I I I MICRONUCLEAR SEGREGATION AT CELL DIVISION IN PAIRS OF SISTER DIVIDERS MICRONUCLEAR COMPLEMENT OF PARENT DIVIDER  MICRONUCLEAR NUMBER OF SISTER DIVIDERS SEGREGATION 1 0 - l a 19-2b -'51-S BETWEEN DIVIDERS  2-2  2-2 2-2  2-2  1-3; 2-2  2-2  1-3; 1-3  2-4  1-3 4-4  2-2  1-3 0-4  1/80(1)  2-2  0-4; 2-2  1/80(1)  2/112(2)  2-2  0-4; 0-4  5/80(6)  1/112(1)  3/3-•3-3  1/80(1)  7/112(6)  3-3  3-3; 2-4  1/80(1)  3-1  3-3 •1-1  1/80(1)  3-3  3-3 ; l - 5  -  1/112(1)  3-3  2-4, 2-4  -  1/112(1)  2-4  2-2 4-4  1/80(1)  3-3  1-5; 1-5  1/80(1)  4-4  4-4 4-4  2/80(3)  -  -  4-4  4-4 ;2-6  2/80(3)  -  -  5-5  5-5 5-5  3-3  •  63/80(79) 81/112(72) 50/50(100)  2/80(3)  -  —  2/112(2) 3/112(3) 1/112(1)  -  -  -  -  1/112(1)  2/112(2)  ' -  2/112(2)  () frequency as a percentage  94 e x t r a m i c r o n u c l e i i n young c e l l s , t o l e s s than two i n o l d e r c e l l s , i n both l i n e s 10-la and 19-2b XIV and  micronuclei  (Tables  XV).  i i ) Temperature E f f e c t s : The 10-la and  e f f e c t of temperature on the phenotype of l i n e s 19-2b  c o u l d not be examined as these  l o s t before a n a l y s i s of the phenotypes was  l i n e s were  completed.  At 27.0°C and 34«5°C m i s d i v i d e r s were found i n c u l t u r e s of the v a r i a n t 2 l - 3 c . low  (between 2-5%).  monsters and  The  frequency  occasionally, s h o r t - l i v e d , heteropolar  E f f e c t s During Nuclear  a)  Autogamy: Nuclear  r e o r g a n i z a t i o n at autogamy was  (approximately  (two m i c r o n u c l e i ) .  exconjugants and  i  was  A l l young c l o n e s had  95%)  a  of normal, v e g e t a t i v e  V a r i a t i o n s i n the numbers of  m i c r o n u c l e i and macronuclear anlagen was  Nuclear  normal i n  No v a r i a t i o n i n n u c l e a r complement  h i g h frequency  doublets.  Reorganization.  observed i n exautogamous c e l l s .  cells  misdividers-was  The m i s d i v i d e r s formed t w o - c e l l  2)  l i n e 10-la.  of  observed i n  exautogamonts of l i n e 19-2b  (Table  events at autogamy were not normal i n l i n e  In a sample of 80 exautogamonts only 46.0% Of the remaining 54.0%, approximately  50.0%  XVI). 21-3c.  were v i a b l e . died within  F i g u r e 19.  The E f f e c t of Age oh the Frequency of Micronuclear  missegregation  10-la and 19-2b. • - • 10-la m-* l9-2b  i n Lines  97  TABLE XIV NUMBER OF MICRONUCLEI AT DIVISION IN CELLS OF DIFFERENT AGES,  LINE 10-la  4-4 3-3 4-0 3-1 2-0 1-1 1-0 0-0  AGE SAMPLE No.ABSIZE NORMAL'  f  10  80  13  16  1  4  3  1  -  1  3  20  100  15  15  2  2  4  1  5  -  1  30  100  18  18  1  1  2  2  3  6  2  4  40  100  20  20  -  -  -  4  4  2  4  -  50  97  25  26  -  -  3  1  4  2  12  3  60  60  14  23  —  —  —  2  6  3  2  1  98  ?TABLE XV NUMBER OF MICRONUCLEI AT DIVISION IN CELLS OF DIFFERENT LINE 1 9 - 2 b  AGES,  AGE SAMPLE No.ABSIZE NORMAL $  4 - 4 5 - 1 4 - 2 3 - 3 4 - 03 - 1 2 - 0 1 - 1 1 - 0 0 - 0  2  -  1  -  -  -  -  5  2  4  2  8  24  7  6  2  26  5  4  10  98  21  21  -  -  1  2  1  5  4  20  100  38  38  -  -  -  2  6  8  6  30  60  22  37  1  1  1  10  -  3  -  40  100  36  36  -  -  -  -  8  10  50  78  50  64  60  60  41  68  -  -  -  -  1  -  -  99  TABLE XVI VARIATION IN THE NUMBER OF NUCLEI IN EXAUTOGAMOUS FIRST CELL CYCLE DIVIDERS, LINE 1 9 - 2 b  MICRONUCLEI  MACRONUCLEAR TOTAL No. No. OF DIVIDING ANLAGEN OF SYNKARYON CELLS PRODUCTS  -2-2  1-1  4(normal)  4-4  1-1  6  2/73 ( 3)  2-6  1-1  6  1/73  4-4  2-2  8  3/73 f 4)  5-5  2-1  8  3/73 ( 4)  4-4  0-0  4  1/73 # 1)  6-6  2-2  10  1/73  ( 1)  2-2  9-9  20  1/73  ( 1)  61/73 (84)*  *() frequency as a percentage  ( 1)  100 two days o f completing autogamy without d i v i d i n g , and approximately 3.0% completed both s i s t e r  one d i v i s i o n , a f t e r which  From a sample o f 100  c e l l s died.  exautogamonts, only 8% had completed  stained  nuclear reorganization  w i t h a normal complement of f o u r n u c l e i , two m i c r o n u c l e i and two macronuclear anlagen.  As many as 47.0% had no  anlagen, and o f those, almost 60.0% had no m i c r o n u c l e i ( P l a t e 10,  B and D).  Of the remaining 52.0% t h a t had  e i t h e r one or two anlagen, 44.0% had e i t h e r a s i n g l e micronucleus or none at a l l .  Even when the normal  n u c l e a r arrangement of two m i c r o n u c l e i and two macron u c l e a r anlagen were p r e s e n t , n u c l e a r morphology was o c c a s i o n a l l y abnormal.  In some cases t h e macronuclear  anlagen were o f very d i f f e r e n t  s i z e s and the macronuclear  fragments appeared t o be s h a t t e r e d i n t o t i n y of chromatin  (Plate 10,A).  splinters  T h i s o b s e r v a t i o n on macro-  n u c l e a r disinte:gr.a#d;bni has a l s o been observed i n amicronucleate Paramecium multimicronucleaturn ( D i l l e r ,  1965).  I n a few cases, exautogamonts c o n t a i n e d many  m i c r o n u c l e i and s m a l l , d a r k l y s t a i n i n g b o d i e s , l a r g e r than m i c r o n u c l e i . are  g r o s s l y underdeveloped  slightly  I t i s p o s s i b l e ' t h a t these bodies macronuclear anlagen  (Plate 10,C).  Exautogamonts c o n t a i n i n g only p a r e n t a l macronuclear fragments d i d not undergo macronuclear r e g e n e r a t i o n . The fragments remained  s m a l l and f a i l e d t o take on t h e c y t o l o g i c a l  101 appearance t y p i c a l of macronuclear  regeneration.  The  c e l l s became t h i n and e v e n t u a l l y d i e d , e i t h e r b e f o r e d i v i s i o n or a f t e r one or two f i s s i o n s Food vacuoles were u s u a l l y absent  ( P l a t e 11 A and B ) .  ( P l a t e 11,B), but  o c c a s i o n a l l y one l a r g e food vacuole was present  b) Conjugation:  ( P l a t e 11,A).  (Line 21-3c only)  P a i r f o r m a t i o n was i r r e g u l a r i n l i n e 21-3c.  Couplets  formed r e a d i l y , but c h a i n s , c l o s e d and open t r i p l e t s , quadruplets and combinations  of these were a l s o formed  (Figure 2 0 ) . Nuclear r e o r g a n i z a t i o n occurred i n most of the c e l l s present i n a clump or c h a i n , but under the circumstances  of t h i s experiment  d i s t i n c t i o n could  not be made between autogamy (cytogamy) and c o n j u g a t i o n . In some cases, members of a clump or c h a i n r e t a i n e d the v e g e t a t i v e n u c l e a r arrangement,- while other members o f the same clump or c h a i n were engaged i n n u c l e a r r e organization.  In any c u l t u r e of c e l l s b e l o n g i n g t o  the l i n e 21-3c many amicronucleate present.  exautogamonts were  When c u l t u r e s c o n t a i n i n g a m i c r o n u c l e a t e s were  mixed w i t h c e l l s o f the opposite mating type, the amicronucleates would r e a d i l y form t y p i c a l mating p a i r s . Since the macronucleus o f the amicronucleates was a l r e a d y fragmented, no f u t h e r n u c l e a r changes was observed amicronucleate  conjugants.  i n the  The normal p a r t n e r o f an  j  102 amicronucleate mating p a i r u s u a l l y underwent the t y p i c a l n u c l e a r changes of c o n j u g a t i o n .  In most cases, both  exconjugants from an amicronuclate s e x u a l arrangement were n o n v i a b l e . p a i r s of 21-3c  Observations on s t a i n e d c o n j u g a t i n g  x w i l d type c e l l s r e v e a l e d two a b n o r m a l i t i e s  i n the n u c l e a r behaviour of the 21-3c  conjugant.  m e i o s i s i n the p a r t n e r s of a mating p a i r was ( P l a t e 12, A and B ) .  asynchronous  The p a r t n e r b e l o n g i n g t o l i n e  always appeared t o be a pregamic w i l d type p a r t n e r .  Firstly,  21-3c  d i v i s i o n behind the  Because of t h i s ,  synchronous,  r e c i p r o c a l exchange of the f e r t i l i z a t i o n p r o n u c l e i d i d not take p l a c e .  Secondly, the p o s i t i o n i n g of the  h a p l o i d m e i o t i c p r o d u c t s was  o f t e n abnormal.  In most  cases, i n the w i l d type conjugant, the m e i o t i c products are of  c e n t r a l l y l o c a t e d i n the conjugant, near the region, the p a r o r a l cone, w h i l e the fragments  macronucleus pronuclei. fragments  of the p r e z y g o t i c  are p e r i p h e r a l , surrounding the h a p l o i d In conjugants b e l o n g i n g t o the l i n e 21-3c, the  of the p r e z y g o t i c macronucleus  throughout the conjugant c e l l ,  are s c a t t e r e d  the p r o n u c l e i seldom l i e  near the r e g i o n of the p a r o r a l cone, but l i k e the are  a l s o s c a t t e r e d about the  cell.  fragments,  I  F i g u r e 20. Anormal Mating C o n f i g u r a t i o n s from a WildnType  Cross t o Line 21-3c  Cells.  I  104  FIGURE 20  105  DISCUSSION  Genetic a n a l y s i s of the two tam mutants, tam A and tam G  ?  r e v e a l s t h a t the abnormal n u c l e a r  events  both a t c e l l d i v i s i o n and at n u c l e a r r e o r g a n i z a t i o n , the changed c e l l morphology and the abnormal morphogenesis of t r i c h o c y s t s are goverened by a s i n g l e , r e c e s s i v e gene i n each mutant.  S e p a r a t i o n of abnormal n u c l e a r  events and abnormal t r i c h o c y s t morphogenesis was not observed  i n the F  2  generation.  The p o s s i b i l i t y  of  the p l e i o t r o p i c e f f e c t o f the tam gene a c t u a l l y  being  caused by two or more c l o s e l y l i n k e d mutations seems u n l i k e l y s i n c e s e v e r a l s i m i l a r mutants have f a i l e d t o show s e p a r a t i o n o f the d i f f e r e n t aspects o f the tam and tarn-like phenotypes (Sonneborn, 1975;  1976).  Ruiz, et a l . ,  In only one of 18 t a m - l i k e mutants i s t h e r e  independent d e t e r m i n a t i o n  of abnormal n u c l e a r  and abnormal t r i c h o c y s t morphogenesis.  traits  These t r a i t s  are s e p a r a t e l y determined i n stock d4-43, which c a r r i e s two separate mutations,  am and nd6 (see Sonneborn, 1975).  106 Even i n am, however, t h e r e i s a p a r t i a l b l o c k a g e trichocyst  discharge  (Aufderheide,  of  personal communication).  D e s p i t e t h i s one p a r t i a l e x c e p t i o n ,  it  i s clear that  t h e r e e x i s t s a c l o s e b i o c h e m i c a l l i n k between c e l l morphology,  trichocyst  morphogenesis and n u c l e a r  morphogenesis.  The phenotype of v a r i a n t l i n e 40-4b (tam G) i n c l u d e d t h e o c c u r r e n c e of l a r g e , b l a c k , crystalline,  vesicular,  cytoplasmic i n c l u s i o n s (spots).  Genetic  a n a l y s i s o f Fg c e l l s from a c r o s s of t h i s s t o c k t o wild-type,  showed independent assortment of t h e tam  phenotype' and t h e spot phenotype i n a 1 : 1 r a t i o . The o r i g i n a l p h e n o t y p e , t h e r e f o r e ,  was produced by  r e c e s s i v e m u t a t i o n s of two s e p a r a t e genes, tam G and s p . Linkage of t h e tamG and sp_ genes appeared u n l i k e l y s i n c e r e c o m b i n a t i o n between them was 4 3 . 0 - 1 0 % ( R i = ' . 5 0 ) .  G e n e t i c a n a l y s i s of v a r i a n t 2 1 - 3 c was not  possible  because c e l l s b e l o n g i n g t o t h i s l i n e d i d not complete conjugation.  L i n e s 1 0 - l a and 19-2b were a c c i d e n t a l l y  l o s t b e f o r e c o m p l e t i o n of the g e n e t i c  analysis.  A comparative a n a l y s i s of c e l l c y c l e l e n g t h s t h a t t h e mean g e n e r a t i o n t i m e s i i n a a l l the v a r i a n t s  shows are  107 l o n g e r than t h a t of w i l d type c e l l s .  The degree t o which  the c e l l c y c l e i s lengthened seems t o correspond t o the degree  of divergence of the v a r i a n t phenotype from the  w i l d type phenotype.  For example, the c e l l c y c l e l e n g t h s  i n the more s e v e r e l y abnormal phenotypes (tam A and tam are extended  by approximately t h r e e hours and two  r e s p e c t i v e l y , w h i l e the v a r i a n t sp, which d i f f e r s slightly  hours only  i n phenotype from w i l d type c e l l s , v. extends  c e l l c y c l e l e n g t h only 20 minutes.  G)  the  Extensions i n c e l l  c y c l e l e n g t h s seem t o be common t o most mutants e x p r e s s i n g non-wild type phenotypes i n Paramecium (see Sonneborn, 1975).  Cell-size, ratio  m a c r o n u c l e a r - s i z e and the l e n g t h - t o - w i d t h  seem t o have been a l t e r e d i n c e l l s homozygous f o r  e i t h e r tam A or tam G.  I n t e r f i s s i o n c e l l s and  dividing  c e l l s were as much as one and one-half times the l e n g t h of w i l d type c e l l s a t the same stage.  T h i s change i n  c e l l s i z e i s not 100$ p e n e t r a n t , but appears c e l l s e x p r e s s i n g the tam phenotype.  only i n  Moreover, tam  A/  tam A c e l l s grown at 17.0°C and/or s t a r v e d f o r s e v e r a l days appear t o r e a d j u s t t o a normal s i z e 120um x 50um).  (approximately  A b s o r p t i o n microspectrophotometric  measurements on Napthol Yellow s t a i n e d G-, c e l l s , r e v e a l s  I  108 t h a t p r o t e i n content i s almost controls.  twice t h a t o f w i l d type  There appears t o be a p o s i t i v e  between c e l l  s i z e and t o t a l p r o t e i n .  This i s i n  agreement w i t h o b s e r v a t i o n s made by Jauker Tetrahymena.  correlation  (1975) i n  The problem o f e x p l a i n i n g t h i s  relatively  s t a b l e d o u b l i n g i n cytoplasmic mass was not r e s o l v e d d u r i n g t h i s study. of  The observed  extention i n the length  the c e l l c y c l e i n tam A/tam A and tam G/tam G c e l l s  could influence c e l l  size.  Jauker  (1975a and b) found  that the gross p r o t e i n content p e r c e l l appeared t o be a f u n c t i o n of the g e n e r a t i o n time, w i t h i n l i m i t s .  He  found t h a t t h e l i n e a r i t y of t o t a l p r o t e i n as a f u n c t i o n of  g e n e r a t i o n time was upset a f t e r very l o n g c e l l c y c l e  lengths.  T h i s o b s e r v a t i o n of Jauker's  could explain  why the double mutant .am/am;tam A/tam A, which has a c e l l c y c l e l e n g t h of f o u r times t h a t of w i l d - t y p e was not c o r r e s p o n d i n g l y e n l a r g e d .  The double mutant  am/am;tam A/tam A. l i k e tam A/tam A. i s only twice the s i z e of w i l d - t y p e c o n t r o l  Since c e l l  must e x i s t .  approximately  cells.  s i z e and macronuclear DNA content  within certain l i m i t s ,  cells,  remain  some type of r e g u l a t i o n process  K i m b a l l (1967)  demonstrated t h a t  inequalities  i n cytoplasmic mass and DNA content, i n t r o d u c e d a t c e l l d i v i s i o n , a r e reduced  over the f o l l o w i n g c e l l  generations,  ' both i n w i l d - t y p e c e l l s and i n c e l l s homozygous f o r am.  109 The  r e l a t i v e l y s t a b l e change i n c e l l and macronuclear  s i z e seen i n tam A/tam A c e l l s does not appear t o be subject  t o r e g u l a t i o n i n the same way.  I t i s therefore  suggested t h a t f a c t o r s other than an extension i n generation  time a r e i n f l u e n t i a l i n p e r p e t u a t i n g  size differences.  One p o s s i b l e e x p l a n a t i o n  these  i s the  occurrence of an i n t e r r u p t i o n i n the normal s t a t e of e q u i l i b r i u m t h a t e x i s t s between c e l l p r o l i f e r a t i o n and c e l l growth r a t e .  This possible s h i f t  i n the e q u i l i b r i u m  would begin when c e r t a i n c h a r a c t e r i s t i c s i n f l u e n c e d by the tam gene come i n t o e x p r e s s i o n .  For example, r a i s i n g  the temperature from 17.0°C t o 27.0°C might an i n c r e a s e  initiate  i n c e l l growth r a t e while c e l l d i v i s i o n r a t e  would not undergo a s i m i l a r i n c r e a s e compensation.  immediately, i n  By the time the r a t e of c e l l d i v i s i o n  catches up w i t h the r a t e of c e l l growth, r e s t o r i n g an e q u i l i b r i u m between the two v a r i a b l e s , the c e l l s have a t t a i n e d a s i z e almost twice that counterparts.  of t h e i r w i l d type  These r e l a t i v e l y s t a b l e changes i n c e l l  s i z e must occur d u r i n g  c e l l d i v i s i o n , s i n c e i t has been  suggested t h a t the number of c o r t i c a l u n i t s p e r c e l l changes only a t t h i s time (D. Jones, 1977).  Interfission  c e l l s may change i n s i z e , but i t might be expected t h a t interfission cell flexibility  s i z e changes are l i m i t e d by the l i m i t e d  i n the s i z e o f the c o r t i c a l u n i t s .  The s t a b l e  110 " change i n macronuclear  DNA  content i s independent  of the  v a r i a t i o n i n t r o d u c e d at c e l l d i v i s i o n through the a c t i o n of  the tam gene.  of  the macronuclear  of  the change i n cytoplasmic mass.  macronuclear  DNA  It' i s suggested t h a t t h i s s t a b l e d o u b l i n g DNA  content i s a d i r e c t  consequence  That the amount of  i s p r o p o r t i o n a l t o the mass of the  has been suggested by Berger  (1977).  This relationship  appears t o be coupled i n a one-way i n t e r a c t i o n and Schmidt; 1977;  cell  Morton and Berger, 1977;  (Berger  Berger,1977).  That i s , changes i n p r o t e i n content do not occur i f DNA  content changes, but i f t o t a l p r o t e i n content changes,  the macronuclear  DNA  content a d j u s t s a c c o r d i n g l y .  one-way c o u p l i n g of cytoplasmic mass and DNA  The  content  r e v e a l s t h a t the n u c l e o - c y t o p l a s m i c r a t i o between them i s not ar-ei?it-ie.al p r e c o n d i t i o n f o r f i s s i o n , as has been suggested  f o r Tetrahymena, (Worthington,  T h i s i s an important  f a c t which can e x p l a i n how  s t a b l e changes i n c e l l growth r a t e without  et a l . ,  relatively  s i z e can occur through changes i n  concommitant r e l a t i v e changes i n the  d i v i s i o n r a t e , which would n e c e s s a r i l y c o r r e c t i n nucleo-cytoplasmic  In  1976).  changes  ratios.  a d d i t i o n t o the r e l a t i v e l y s t a b l e change i n c e l l  s i z e and mean macronuclear  DNA  content i n tam A/tam A  Ill c e l l s , f u r t h e r changes i n DNA content are i n t r o d u c e d at  fission  through the a c t i o n of the tam gene.  changes are not r e l a t i v e l y  These  s t a b l e , but appear t o be  subject t o r e g u l a t i o n (Berger and Schmidt, 1977).  It  has been found t h a t t h e v a r i a n c e o f induced n u c l e a r DNA content from the p o p u l a t i o n mean, i s reduced by one h a l f i n a s i n g l e c e l l c y c l e , and t h a t the v a r i a n c e continues t o be reduced by one h a l f i n each s u c c e s s i v e c e l l cycle  (Berger and Schmidt, 1977).  Although the mean c e l l c y c l e l e n g t h i n l i n e 2 l - 3 c i s almost f o u r times the l e n g t h o f t h a t o f w i l d type c e l l s , the average  c e l l s i z e i s equal t o or s m a l l e r  than t h a t o f w i l d - t y p e c e l l s . to  T h i s can be a t t r i b u t e d  the f a c t that the m a j o r i t y (83.0%)  c u l t u r e o f 21-3c  of c e l l s  i n any  are amicronucleate exautogamonts.  Feeding appears t o be a major problem T h i s s u g g e s t i o n i s based  i n such  cells.  on o b s e r v a t i o n s on s t a i n e d  cells  which r e v e a l an absence of food-vacuoles i n the m a j o r i t y of  amicronuclate c e l l s .  These amicronuclate c e l l s  p e r s i s t f o r a few days, but few f a i l t o complete c e l l cycle.  may  an e n t i r e  Replacement o f the o r a l apparatus and  v e n t r a l c i l i a t i o n a p p a r e n t l y occurs at c o n j u g a t i o n , i n Paramecium (Roque, 1956).  I t has been suggested  that  112  replacement  o f t h e o r a l apparatus at c o n j u g a t i o n , i n  amicronucleate c e l l s , i s incomplete Skoblo  (1969)  1965).  found t h a t amicronucleate Paramecium  caudatum exconjugants Tavrovskaya  (Diller,  (1969)  c o u l d not f e e d , and Ossipov and  found that amicronucleate  exconjugants  showed no s i g n s of forming a new g u l l e t i n P. caudatum. I t i s p o s s i b l e , t h e r e f o r e , t h a t r e g e n e r a t i o n of the o r a l apparatus i n P_. t e t r a u r e l i a i s incomplete i n amicronucleate exconjugants.  Macronuclear  d i v i s i o n i n c i l i a t e s i s unusual i n  t h a t 1 ) t h e macronuclear out k a r y o k i n e s i s ,  envelope remains  through-  2 ) a m i t o t i c apparatus i s not present  i n t h e d i v i d i n g macronucleus and condensation o f chromosomes. accomplished  intact  3 ) t h e r e i s no  Macronuclear  division i s  through simple c o n s t r i c t i o n o f the macro-  nucleus i n t o approximately two equal p a r t s .  Although  a t y p i c a l m i t o t i c s p i n d l e i s not present i n the d i v i d i n g macronucleus,  i n t r a n u c l e a r m i c r o t u b u l e s , and bundles o f  m i c r o t u b u l e s a s s o c i a t e d w i t h t h e n u c l e a r membrane have been observed i n many c i l i a t e s 1969;  (Tucker, 1 9 6 7 ; Suganuma,  Roth and M i n i c k , 1 9 6 1 ; Tamura, ejp a l . , 1 9 6 9 ;  Jurand and Selman,  1970;  Inaba and Kudo,  1972).  the many c y t o l o g i c a l o b s e r v a t i o n s on a m i t o t i c a l l y  Despite dividing  113 macronuclei, the mechanisms governing the morphogenetic events are not known.  These morphogenetic events are  s e v e r e l y a l t e r e d i n c e l l s e x p r e s s i n g the tam phenotype. The m a c r o n u c l e i o f tam A/tam A and tam G/tam G c e l l s undergo the very e a r l y p r e d i v i s i o n events and reach a c e n t r a l p o s i t i o n i n the p r e d i v i s i o n c e l l .  However, the  macronuclei i n these c e l l s f o r g o any f u r t h e r morphog e n e s i s , they f a i l t o reach the normal s u b c o r t i c a l on the d o r s a l s i d e o f the c e l l ,  location  they f a i l t o elongate  p r o p e r l y and macronuclear c o n s t r i c t i o n does not take place.  T h i s i n a b i l i t y o f the macronucleus t o complete  p r e d i v i s i o n morphogenesis has been observed i n other tam mutants, n o t a b l y tam 6, tam 8 (Beisson and R o s s i g n o l , and tam 38 (Ruiz, e t a l . , 1976).  1975)  There are s e v e r a l p o s s i b l e  e x p l a n a t i o n s t h a t c o u l d account f o r the abnormal behavior of the macronucleus i n homozygous tam c e l l s . the  F a i l u r e of  macronucleus t o complete p r e d i v i s i o n morphogenesis  may be a t t r i b u t e d t o : of the macronucleus;  1) improper cytoplasmic  localization  2) improper p o l y m e r i z a t i o n of i n t r a -  n u c l e a r and/or e x t r a n u c l e a r m i c r o t u b u l e s and membrane attachment  3) d e f e c t i v e  sites.  A s e r i e s o f g r a f t i n g experiments on S t e n t o r l e n d s support t o the f i r s t ,  '  possible explanation.  These  \  114 experiments  r e v e a l t h a t c e r t a i n a s p e c t s o f macronuclear  behavior are i n f l u e n c e d by c o r t i c a l s i g n a l s (de T e r r a ,  1971 and 1973).  The c o r t i c a l s i g n a l s have a very short  range of i n f l u e n c e and, t h e r e f o r e , can o n l y a c t on s u b c o r t i c a l l y l o c a t e d macronuclei  1973).  (de T e r r a , 1971 and  I f such a system of c o r t i c a l i n f l u e n c e e x i s t s  i n Paramecium, and i f ,  as i n S t e n t o r , these s i g n a l s have  a very short range, then, i t may be suggested,  t h a t the  misplaced macronucleus f a i l s t o r e c e i v e the s i g n a l s t h a t morphogenesis should  proceed.  S e v e r a l r e f e r e n c e s l e n d support t o the second p o s s i b i l i t y , t h a t f u n c t i o n a l l y inadequate  microtubules  may be the cause of the f a i l u r e of the macronucleus t o continue w i t h p r e d i v i s i o n morphogenesis, such as the f a i l u r e t o elongate.  Tamura, e_t aJL., (1969) found t h a t  the macronuclei o f c e l l s t r e a t e d w i t h c o l c h i c i n e , a drug known t o i n t e r f e r e w i t h microtubule p o l y m e r i z a t i o n f a i l e d t o elongate p r o p e r l y :  improper  placement of the  macronucleus was observed, and, i n c e l l s t h a t  completed  n u c l e a r d i v i s i o n , the d i v i s i o n of the macronucleus was unequal.  W i l l i a m s and W i l l i a m s (1976) r e p o r t e d t h a t  t r e a t e d w i t h c o l c h i c i n e j u s t p r i o r t o macronuclear e l o n g a t i o n , completed  d i v i s i o n , but they found t h a t  cells  115 macronuclear  morphogenesis was abnormal.  i n treated c e l l s ,  The m a c r o n u c l e i  f a i l e d to elogate, nuclear c o n s t r i c t i o n  d i d not occur, the m a c r o n u c l e i remained  i n a central  l o c a t i o n and n u c l e a r morphology was abnormal.  Electron  m i c r o s c o p i c examination o f these c o l c h i c i n e t r e a t e d r e v e a l e d a complete  absence o f the i n t r a n u c l e a r and  membrane a s s o c i a t e d m i c r o t u b u l e s .  Ruiz, .et a l . ,  r e p o r t e d the i n d u c t i o n of phenocopies tam  cells  (1976)  of the mutant  38 by t r e a t i n g w i l d - t y p e c e l l s w i t h e i t h e r  colchicine  or v i n b l a s t i n .  F u r t h e r evidence f o r the p o s s i b l e involvement o f m i c r o t u b u l e s i n macronuclear  e l o n g a t i o n and c o n s t r i c t i o n  comes from a study by Walker and Goode (1976) on the r o l e o f m i c r o t u b u l e s i n macronuclear  d i v i s i o n i n two  h y p o t r i c h s , G a s t r o s t y l a and S t y l o n y c h i a .  The authors  found t h a t the macronuclei of c e l l s t r e a t e d w i t h deutrium oxide, an agent  60.0%  known t o hyperpolymerize  m i c r o t u b u l e s , were hyperextended,  e x h i b i t e d abnormal  morphology, and o f t e n f a i l e d t o complete k a r y o k i n e s i s .  However, agents other than those d i r e c t l y m i c r o t u b u l e assembly,  affecting  can a l s o induce s i m i l a r , abnormal  events i n ^PK©<iivision macronuclear  morphogenesis.  t r e a t e d p r i o r t o , or a t i n t e r f i s s i o n age 0 . 7 ,  with  Cells  116 actinomycin D (a drug known t o i n h i b i t RNA f a i l to divide  (Gill  and Hanson, 1 9 7 6 ) . 0.7  a f t e r i n t e r f i s s i o n age  completed  i n many cases c y t o k i n e s i s was  synthesis)  Cells treated  cytokinesis,  but  not accompanied by  k a r y o k i n e s i s - the i n d i v i d u a l p r e f i s s i o n macronucleus was  r e t a i n e d i n the p r o t e r .  G i l l and Hanson (1976)  a l s o found t h a t , where c y t o k i n e s i s was  accompanied by  k a r y o k i n e s i s , i n actinomycin D t r e a t e d c e l l s , macron u c l e a r d i v i s i o n was s m a l l e r amount of DNA  unequal  and c e l l s r e c e i v i n g the  were i n v i a b l e .  Examination  of the  actinomycin D t r e a t e d c e l l s r e v e a l e d l a r g e aggregates of  p r o t e i n - l i k e m a t e r i a l which G i l l and Hanson (1976)  suggested may  be r e l a t e d t o microtubule  formation.  The authors d i d not o f f e r an e x p l a n a t i o n as t o actinomycin D was  how  a f f e c t i n g microtubule p o l y m e r i z a t i o n .  The p o s s i b i l i t y remains t h a t any t o x i c drug i n t e r f e r e w i t h p r e f i s s i o n macronuclear  will  morphogenesis .  There i s a suggestion, however, t h a t a c t i n o m y c i n D i n h i b i t s the t r a n s c r i p t i o n of a microtubule factor  assembly  (Bierber, 1972).  Although w i l d - t y p e c e l l s , t r e a t e d w i t h a n t i - m i c r o t u b u l e drugs mimick the phenotype produced mutations,  i t does not mean t h a t the tam  by  tam  lesion  directly  117 a f f e c t s the microtubule p o p u l a t i o n .  The  ability  of m i c r o t u b u l e s t o polymerize i n homozygous tam c e l l s i s c l e a r l y demonstrated by the a b i l i t y of these c e l l s t o r a p i d l y regenerate shed  cilia.  In a d d i t i o n , i t i s d i f f i c u l t t o imagine what p o s s i b l e e f f e c t m i c r o t u b u l e s would have i n i n f l u e n c i n g  trichocyst  morphogenesis.  The t h i r d p o s s i b l e e x p l a n a t i o n i s t h a t the  tam  l e s i o n a f f e c t s the plasma membrane and/or the macron u c l e a r envelope. mutation  One  p o s s i b i l i t y i s t h a t the  tam  l e a d s t o a d i s r u p t i o n of intramembraneous  p a r t i c l e a r r a y s , which would obscure  attachment-sites  normally a v a i l a b l e t o the p r e f i s s i o n macronucleus. I f membrane attachment s i t e s are abnormal i n homozygous tam A and tam G c e l l s , then the i n a b i l i t y of m i c r o t u b u l e s (or m i c r o f i l a m e n t s ) t o make an attachment would be compatible w i t h the apparent microtubules,  incompetence of the  and the a b i l i t y of a n t i - m i c r o t u b u l e  drugs t o mimick the tam phenotype.  The p o s s i b i l t y of  a d i s o r d e r e d membrane e x i s t i n g i n homozygous tam  cells  can more r e a d i l y be a p p l i e d as the basiespsrobil-em'*underl y i n g the p l e i o t r o p i c e f f e c t  of the tam  gene.  Intra-  membraneous microtubule b r i d g e s are known t o e x i s t  (see  118 Allen, 1975).  In a d d i t i o n t h e r e i s evidence t o support  the i d e a t h a t m i c r o t u b u l e s a r e i n v o l v e d i n membranebound, i n t r a c e l l u l a r p a r t i c l e movement (such as the Holmes and Choppin, 1 9 6 8 ;  r e l o c a l i z a t i o n of a nucleus. M e s s i e r and A u c l a i r , 1 9 7 3 ;  Robison and C h a r l t o n , 1 9 7 3 ;  Wagner and Rosenberg, 1 9 7 3 ;  A l l e n , 1 9 7 4 ) , and evidence  t h a t supports the i d e a o f e x t r a n u c l e a r microtubule a s s o c i a t i o n w i t h the macronuclear ciliates  (Roth and Shigenaka,  J e n k i n s , 1968;  envelope  i n many  1964; Schulster, 1965;  Inaba and Sotokawa, 1 9 6 8 ) .  I t may be  suggested, t h e r e f o r e , t h a t i n the w i l d - t y p e  cell,  •microtubule l i n k a g e s form between the plasma membrane and the macronuclear  envelope, g u i d i n g the macronucleus  to a s u b c o r t i c a l l o c a t i o n and f i r m l y anchoring the p r e f i s s i o n macronucleus t o the c o r t e x , i n p r e p a r a t i o n f o r macronuclear  elongation.  Thus one c o u l d s p e c u l a t e  t h a t these microtubule l i n k a g e s f a i l t o form i n homozygous tam c e l l s due t o the i n a b i l i t y of the microt u b u l e s t o r e c o g n i z e the a l t e r e d membrane sites.  Consequently,  attachment-  r e l o c a t i o n o f the p r e f i s s i o n  macronucleus and f u r t h e r morphogenesis does not take place.  Experimental support f o r t h i s i d e a exists i n  the l i t e r a t u r e p e r t a i n i n g t o t r i c h o c y s t morphogenesis and t r i c h o c y s t attachment,  and w i l l be d i s c u s s e d l a t e r .  Since the degree of macronuclear m i s s e g r e g a t i o n v a r i e d between d i f f e r e n t experiments, the p o s s i b l e i n f l u e n c e s of age and temperature on penetrance and e x p r e s s i o n of the tam gene were examined.  Nobili  (1961)  had found t h a t a s i m i l a r mutant, am/am, showed agedependent  expression.  Only homozygous tam A  were examined f o r age-dependent  cells  e x p r e s s i o n . When a l l  the aspects of the tam gene were c o n s i d e r e d t h e r e was age-dependent  no  e x p r e s s i o n i n tam A/tam A i n d i v i d u a l s .  However, complete macronuclear m i s s e g r e g a t i o n , when examined alone, showed a d e f i n i t e i n c r e a s e i n frequency with i n c r e a s i n g c l o n a l age.  At the same time, p a r t i a l  macronuclear m i s s e g r e g a t i o n showed a decrease i n frequency w i t h i n c r e a s i n g age of the c l o n e .  Thus, i t  appeared t h a t the more severe form of macronuclear missegregation increased i n older clones.  E x p r e s s i o n of mutations of both tam A and tam G showed a s l i g h t temperature dependency.  Macronuclear  m i s s e g r e g a t i o n i n c r e a s e d i n frequency at 27.0°C when compared w i t h the frequency at 34.5°C.  I f microtubule  p o l y m e r i z a t i o n i s the b a s i c problem behind macronuclear morphogenesis,  abnormal  then one c o u l d s p e c u l a t e  that the r a t e of m i c r o t u b u l e p o l y m e r i z a t i o n might  be  120 i n c r e a s e d at the higher temperature. suggested  I t c o u l d a l s o be  t h a t changes i n membrane-bound p a r t i c l e  a r r a y s occur at the h i g h e r temperature, possible attachment-sites. may  thus unmasking  A l t e r n a t i v e l y , the tam  genes  be c o l d s e n s i t i v e , g e n e s with impaired f u n c t i o n at  lower  temperatures.  Abnormal d i s t r i b u t i o n of m i c r o n u c l e i at c e l l  division  occurs both i n c e l l s e x p r e s s i n g the tam phenotype  and  i n c e l l s b e l o n g i n g t o the l i n e s 10-la-and  From  19-2b.  c y t o l o g i c a l o b s e r v a t i o n s on s t a i n e d c e l l s , i t appears > t h a t the m i c r o n u c l e i f a i l t o migrate t o t h e i r normal l o c a t i o n at the p o l e s of the d i v i d i n g c e l l .  Instead,  the m i c r o n u c l e i remain c l u s t e r e d around the plane cleavage and are randomly d i s t r i b u t e d by the f i s s i o n furrow.  of  advancing  T h i s f r e q u e n t l y l e a d s t o e r r o r s i n the  d i s t r i b u t i o n of m i c r o n u c l e i at c e l l d i v i s i o n .  Improper  cytoplasmic l o c a l i z a t i o n appears t o be the b a s i s of both m i c r o n u c l e a r and macronuclear abnormal p a t t e r n s i n homozygous tam  cells.  behaviour  The f a c t t h a t other  tam mutants a l s o e x h i b i t i r r e g u l a r d i s t r i b u t i o n of m i c r o n u c l e i would suggest between the two  the p o s s i b i l i t y of a c o u p l i n g  types of n u c l e a r behaviour  (see Ruiz, et a l . , 1 9 7 6 ) .  traits  However, i f some s o r t of  121 c o u p l i n g does e x i s t , i t cannot be very t i g h t , irregular distributions  since  o f m i c r o n u c l e i can occur i n  the absence of macronuclear missegregation  (lines  10-la and 19-2b).  Two hypotheses are suggested t o e x p l a i n the observed misplacement of m i c r o n u c l e i i n tam A/tam A and tam G/tam G c e l l s .  F i r s t l y , misplacement of the  m i c r o n u c l e i may a r i s e from the f a i l u r e of the m i t o t i c s p i n d l e t o elongate Ruiz, e t _ a l . , 1976).  (a p o s s i b i l i t y a l s o suggested by Secondly, micronuclear  mis-  placement may be due t o the f a i l u r e o f membrane or microtubule  l i n k a g e s t o form between the macronucleus  and the m i c r o n u c l e i . mitotic  bridges  In £ . t e t r a u r e l i a , the i n t r a n u c l e a r  s p i n d l e forms a s e p a r a t i o n s p i n d l e , pushing the  daughter m i c r o n u c l e i away from the l a t i t u d e ;' towrds the p o l e s o f the d i v i d i n g  cell  of cleavage,  (see Tucker, 1967).  Thus, inadequate e l o n g a t i o n of the s e p a r a t i o n could r e s u l t  spindle  i n the l a c k of movement of the daughter  m i c r o n u c l e i and the subsequent abnormal d i s t r i b u t i o n of the m i c r o n u c l e i a t f i s s i o n . the l i t e r a t u r e t o support  There i s l i t t l e  evidence i n  t h e second p o s s i b i l i t y .  Only  a few authors have observed macronuclear-micronuclear linkages during nuclear d i v i s i o n .  The f i r s t  observation  122 of t h i s k i n d was made by Tucker c i l i a t e Nassula.  (1967) i n the  The form o f l i n k a g e i n t h i s s p e c i e s  c o n s i s t s o f membrane b r i d g e s connecting the d i v i d i n g macronucleus w i t h the m i t o t i c a l l y d i v i d i n g m i c r o n u c l e i . Tucker suggests t h a t the b r i d g e s may be important i n a s s u r i n g even d i s t r i b u t i o n o f m i c r o n u c l e i t o s i s t e r  cells.  He. went on t o s p e c u l a t e t h a t i f these membrane b r i d g e s were absent and the daughter m i c r o n u c l e i were f r e e t o move through the cytoplasm, they might be u n e q u a l l y d i s t r i b u t e d between s i s t e r c e l l s when c y t o k i n e s i s was completed.  In Paramecium multimicronucleatum a d i f f e r e n t  type o f macronuclear-micronuclear l i n k a g e has been observed  (Inaba and Kudo, 1972).  In t h i s l i n k a g e system  m i c r o t u b u l e s a r e found t o connect the e a r l y  dividing  macronucleus w i t h the m i t o t i c a l l y d i v i d i n g m i c r o n u c l e i . However, the m i c r o t u b u l e s a r e not seen a f t e r m i c r o n u c l e a r metaphase.  The suggested r o l e f o r t h i s m i c r o t u b u l e l i n k  between the macronucleus and the micronucleus, i s t h a t the m i c r o t u b u l e s separate the m i c r o n u c l e i from the macronucleus d u r i n g the e a r l y stage o f macronuclear  division.  In Paramecium b u r s a r i a , extranucleaas m i c r o t u b u l e s have been seen t o connect the micronucleus t o the c e l l s u r f a c e d u r i n g metaphase and through anaphase o f m i c r o n u c l e a r division  (Lewis, et a l . ,  1976).  The authors d i d not  123 d i s c u s s the p o s s i b l e s i g n i f i c a n c e of t h i s  observation,  but the p o s s i b i l i t y s t i l l remains t h a t these l i n k a g e s may  microtubule  account f o r equal d i s t r i b u t i o n of m i c r o n u c l e i  t o daughter n u c l e i a t  fission.  T h e o r e t i c a l l y , i r r e g u l a r micronuclear could e v e n t u a l l y give r i s e t o c e l l s w i t h (10, 16 and many more), and have been observed i n any  distribution multi-micronuclei  yet no more than s i x m i c r o n u c l e i  one  interfission cell.  suggests e i t h e r t h a t c e l l s w i t h a very h i g h complement have reduced v i a b i l i t y ,  This  micronuclear  or t h a t some s o r t of  i n t r a c e l l u l a r " r e g u l a t i o n of micronuclear  number e x i s t s .  A type of r e g u l a t i o n of m i c r o n u c l e a r  number e x i s t s i n  Gastrostyla s t e i n i i .  observed t h a t  Walker (1976)  a l l f o u r m i c r o n u c l e i took p a r t i n m i t o s i s at any c e l l d i v i s i o n or at excystment.  not  given  He found there was  an  i n t r a c e l l u l a r d i s c r i m i n a t i o n between the m i c r o n u c l e i t h a t one  or more may  forgo d i v i s i o n .  T h i s asynchronous  behaviour of m i c r o n u c l e i i n G a s t r o s t y l a may r e g u l a t i n g micronuclear  numbers.  be away of  Since numbers of  m i c r o n u c l e i have been observed i n s t a i n e d cells-, are not m u l t i p l e s of two  and  and, t h a t there e x i s t s an  that age  dependent s h i f t toward lower numbers of m i c r o n u c l e i , the p o s s i b i l i t y remains t h a t P_. t e t r a u r e l i a can c o n t r o l  124 the number of m i c r o n u c l e i any  one  cell  The  t r i c h o c y s t s of homozygous tam  and  c e l l s are  a t t a c h e d t o the  unable  cell's  they have an abnormal morphology ( f o o t b a l l -  shaped i n tam The  in  cycle.  t o d i s c h a r g e , they are not surface  embarking on m i t o s i s  A/tam A c e l l s and  stubby i n tam  G/tam  G).  normal t r i c h o c y s t c y c l e begins w i t h morphogenesis  inside small, organelle  cytoplasmic v e s i c l e s .  then migrates t o the  The  f u l l y formed  c e l l ' s c o r t e x where i t  a t t a c h e s at s p e c i f i c s i t e s on the plasma membrane. Exo.cytosis i s the Both genes, tam  f i n a l event i n the t r i c h o c y s t  A and  very e a r l y i n the  tam  cycle.  G,  affect trichocyst  Detailed  shaped t r i c h o c y s t s r e v e a l that they may  few  within  the  development  examination of f o o t b a l l -  a number are t i p l e s s , but  have unattached, a b o r t i v e  organelle,  cycle.  t i p s l y i n g beside  v e s i c l e (Pollack,  1974).  the  A very  have misshapen t i p s , t h a t are a t t a c h e d ( P o l l a c k ,  1974).  Stubby t r i c h o c y s t s have a v a r i a b l e morphology, a few be a t t a c h e d t o the of the  t i p and  side the tam  and  c e l l ' s cortex; i n some, a s m a l l segment  some sheath m a t e r i a l  organelle  may  (Pollack,  1974).  are  located  along  In twelve other  tarn-like mutants, abnormal t r i c h o c y s t morphogenesis  i s apparent i n a d d i t i o n t o abnormal n u c l e a r behaviour.  125 In f i v e other mutants, nd 6 , nd 9, nd 3. nd 7 and p t 2 , (Ruiz, e t _ a l . , 1976) i n which t r i c h o c y s t s f a i l t o d i s c h a r g e , t h e r e i s no evidence  of abnormal n u c l e a r  behaviour.  In these f i v e l a t t e r mutants,  trichocysts  are capable  of attachment t o the plasma membrane, whereas  i n a l l the other s i m i l a r mutants, where both and n u c l e a r t r a i t s are i n evidence trichocyst  t o g e t h e r , no  attachment i s observed.  On the b a s i s of the  apparent c o r r e l a t i o n between t r i c h o c y s t and n u c l e a r behaviour,  trichocyst  R u i z , .et _ a l .  attachment  (1976)  suggest  t h a t the n u c l e a r a b n o r m a l i t i e s common t o t h e i r tam mutants a r e d i r e c t l y due t o the absence o f a t t a c h e d trichocysts.  They f u r t h e r  with unattached  trichocysts  nuclear d i v i s i o n s .  suggest,  t h a t a l l mutants  should have abnormal  T h i s theory, put forward  et a l . (1976) would f i t w i t h the i d e a t h a t  by Ruiz,  trichocyst  and n u c l e a r morphogenesis a r e l i n k e d t o the same b i o c h e m i c a l pathway, or comprise two major branches of one pathway. I f t h i s were the case t h e t r i c h o c y s t branch would be d i s t a l t o the n u c l e a r branch.  Thus, one c o u l d  independent t r i c h o c y s t mutations,  anticipate  but n u c l e a r a b n o r m a l i t i e s  would always be a s s o c i a t e d with t r i c h o c y s t  abnormalities.  The most d i f f i c u l t p a r t o f d i s c u s s i n g the tam gene i s f i n d i n g a common denominator between the t r i c h o c y s t  126 trait  and the n u c l e a r t r a i t .  It i s d i f f i c u l t  imagine t h a t a v i t a l i n c l u s i o n such as the  to  nucleus,  ^should be so t i g h t l y coupled t o a system of o r g a n e l l e s , the t r i c h o c y s t s , l a c k i n g any  obvious f u n c t i o n .  The  r e l a t i o n s h i p might be very s u b t l e or i t might be  something  as obvious as m i c r o t u b u l e s , m i c r o f i l a m e n t s or membrane attachment s i t e s .  T h i s study p r o v i d e s no e x p l a n a t i o n of  t h e . p l e i o t r o p i c nature  of the tam  gene.  The most  p l a u s i b l e suggestion i s t h a t membrane-bound p a r t i c l e . a r r a y s may  be abnormal i n c e l l s e x p r e s s i n g the tam  phenotype.  T h i s c o u l d then r e s u l t i n the l a c k of f o r m a t i o n of r e c o g n i z a b l e attachment s i t e s f o r t r i c h o c y s t s as w e l l as m i c r o t u b u l e s .  I f t h i s were the case, one  could  a n t i c i p a t e t h a t o r g a n e l l e movement would be impaired, which i s the b a s i c problem observed H cells.  In support  i n homozygous tam A and  of t h i s idead i s the f a c t t h a t  tam  specific  plasma membrane s i t e s have been i d e n t i f i e d f o r t r i c h o c y s t and i n Tetrahymena, mucocyst attachment. ;  The  attachment s i t e s are s e v e r l y a l t e r e d i n t l ,  trichocyst  tam  8 and  nd_9 (Beisson, et a l . , 1976). Tr'lchocyst membrane attachment s i t e s appear as an ordered a r r a y of p a r t i c l e s i n an i n n e r r o s e t t e and an outer r i n g S a t i r , _ e t a l . , 1972; et_al.,  1976).  (Janish,  P l a t t n e r , et a l . ,  1973;  arranged 1972; Beisson,  Beisson et a l . - (1976) found t h a t the  r i n g of p a r t i c l e s i s always present, even i n mutants  outer  127 containing  no t r i c h o c y s t s  ( t l ) . In mutants  containing  unattached t r i c h o c y s t s or no t r i c h o c y s t s , the outer r i n g of p a r t i c l e s i s c o l l a p s e d ,  forming a p a r e n t h e s i s .  In  c e l l s w i t h a t t a c h e d t r i c h o c y s t s , the outer r i n g s a r e c i r c u l a r (nd9  and w i l d t y p e ) .  collapsed  The authors suggest t h a t the  r i n g s correspond t o u n f i l l e d  trichocyst  attachment s i t e s while the c i r c l e s correspond t o filled  sites.  In a d d i t i o n ,  Beisson, et a l . (1976)  found t h a t the i n n e r r o s e t t e s  a r e only present when  t r i c h o c y s t s a r e a t t a c h e d , and t h a t they appear t o be an e s s e n t i a l f e a t u r e  o f t r i c h o c y s t d i s c h a r g e ( i n nd9  at 27.0°C, there i s t r i c h o c y s t attachment but no d i s c h a r g e ; the r o s e t t e s  a r e abnormal. At the p e r m i s s i v e  temperature^ the r o s e t t e s are able t o d i s c h a r g e ) .  r e c o v e r and the t r i c h o c y s t s An important p o i n t made by the  authors i s t h a t t r i c h o c y s t attachment a p p a r e n t l y b r i n g s about p a r t i c l e rearrangement i n the plasma membrane. Ruiz, ejt a l . (1976) take t h i s p o i n t  further,  suggesting  t h a t t r i c h o c y s t attachment may t r i g g e r other changes i n membrane o r g a n i z a t i o n ,  and t h a t these attachment-dependent  changes may be important f o r membrane-nucleus- i n t e r a c t i o n s . T h i s would e x p l a i n the t i g h t c o u p l i n g  between t r i c h o c y s t  attachment and n u c l e a r behaviour a t c e l l d i v i s i o n .  128 Recent  experimental evidence suggests that the  l e s i o n s due t o the tam mutations may be d i r e c t l y  affecting  the t r i c h o c y s t s themselves, r a t h e r than the sequence of m i g r a t i o n and attachment  of the t r i c h o c y s t s .  Evidence  f o r t h i s came from a study employing m i c r o i n j e c t i o n o f cytoplasm c o n t a i n i n g t r i c h o c y s t s , i n JP. t e t r a u r e l i a (Aufderheide, 1977).  Aufderheide  (1977) used f t A c e l l s  ( f o o t b a l l - s h a p e d t r i c h o c y s t s that w i l l not d i s c h a r g e ) as the host c e l l and i n j e c t e d cytoplasm c o n t a i n i n g m o t i l e , w i l d type t r i c h o c y s t s .  He found t h a t w i t h i n ©ne hour  the w i l d type t r i c h o c y s t s had a l i g n e d and a t t a c h e d , and were capable o f d i s c h a r g e , i n the f o r e i g n  cytoplasm.  However, i n j e c t i o n of cytoplasm c o n t a i n i n g t r i c h o c y s t s of nd A or tam 8 i n f t A host c e l l s , r e v e a l e d t h a t these t r i c h o c y s t s remained  nonmotile and i n c a p a b l e of d i s c h a r g e .  W i l d type t r i c h o c y s t s i n t r o d u c e d i n t o nd A or tam 8 host c e l l s remained m o t i l e and were capable of d i s c h a r g e . T h i s study demonstrates  that the mutations, nd A and tam 8,  appear t o d i r e c t l y a f f e c t the t r i c h o c y s t i t s e l f .  This  would agree t o a c e r t a i n extent w i t h the p o s s i b i l i t y of a membrane d i s o r d e r b e i n g the common l i n k i n the p l e i o t r o p i c e f f e c t of the tam gene.  Changes i n the p e r m e a b i l i t y o f c e l l membranes ( o r o r g a n e l l e membranes) can r e s u l t i n s i z e and shape changes  129 (see K a s t u r i B a i and Maujuba, 1977)  and must r e s u l t i n  changes i n intramembraneous p a r t i c l e arrangement. l a t t e r changes w i l l i n t e r f e r e with microtubule i n t e r a c t i o n s and p o s s i b l y o r g a n e l l e movement. a l l the experimental  .  These  (microfilament) Thus,  evidence t o date i s c o n s i s t e n t  with  the p o s s i b i l i t y t h a t the tam mutation i s d i r e c t l y a f f e c t i n g the membrane system i n homozygous tam  A phenomenon evident the f o r m a t i o n  i n both tam  cells.  G and  tam  38 i s  of e x t r a m i c r o n u c l e i and macronuclear anlagen  a t n u c l e a r r e o r g a n i z a t i o n , a phenomenon not observed i n tam  A.  and tam  T h i s s i m i l a r i t y i n phenotype between tam 38 i s s u r p r i s i n g s i n c e tam  while both tam The  38 and tam  possible existence  G has  G  stubby t r i c h o c y s t s  A have f o o t b a l l - s h a p e d t r i c h o c y s t s .  of n u c l e u s - c o r t i c a l attachments  d u r i n g d i f f e r e n t i a t i o n o f the synkaryon d i v i s i o n p r o d u c t s remains unexplored.  However, Sonneborn (1954) c l e a r l y  demonstrated t h a t the synkaryon p r o d u c t s move t o a r e g i o n of the r e o r g a n i z i n g c e l l , normal d i f f e r e n t i a t i o n . cytoplasmic  regions  i n order t o undergo  T h i s movement towards s p e c i f i c  ( a n t e r i o r and p o s t e r i o r p o l e s  the r e o r g a n i z i n g c e l l ) probably microtubules,  specific  i n v o l v e s guidance from  and t h i s guidance may  c o r t i c a l membrane l i n k a g e .  of  require nucleus-  i  I t i s p o s s i b l e t h a t when the  ,130 synkaryon comes i n t o a s p e c i f i c a r e a i n the c e l l , i t receives signals f o r d i v i s i o n .  In the case o f tam G,  then, improper s p a t i a l p o s i t i o n i n g of the synkaryon may l e a d t o i n a c c u r a c i e s both i n d i v i s i o n and i n d i f f e r e n t i a t i o n of ,the synkaryon p r o d u c t s .  In c o n c l u s i o n ,  i t would appear t h a t proper  cytoplasmic placement nuclei  of the macronucleus, the m i c r o -  and t h e d i v i s i o n products o f the synkaryon  i s essential dividing  f o r normal n u c l e a r behaviour w i t h i n the  or r e o r g a n i z i n g  arrangements  cell.  I suggest t h a t  of membrane-bound p a r t i c l e s ,  abnormal  or changes  i n membrane p e r m e a b i l i t y may be the b a s i s of the tam A and tam G phenotypes.  L  BIBLIOGRAPHY  Adoutte, A., and J . B e i s s o n . (1970) Cytoplasmic i n h e r i t a n c e of erythromycin r e s i s t a n t mutations i n Paramecium. Mol. and Gen. Genet, vol:106% 70-77. Alexander, P., J.T. L e t t and G.Mi P a r k i n s . (1961) Instability of a l k y l a t e d d e o x y r i b o n u c l e i c a c i d i n r e l a t i o n t o t h e mechanism of chemical mutagenesis. Biochim. Biophys. A c t a  vol:48,  423.  A l l e n , S., and I . Gibson. 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Rec.  vol:78. 53-54.  Sonneborn, T.M. (1947) Recent advances i n the g e n e t i c s of Paramecium and E u p l o t e s . Adv. Genet, v o l : 1 , 263-358. Sonneborn, T.M. (1953) Environmental c o n t r o l o f the d u r a t i o n o f phenomic o r cytoplasmic l a g i n Paramecium a u r e l i a . M i c r o b i o l . Genet. B u l l , v o l : 7 , 23 Sonneborn, T.M. (1953) P a t t e r n s o f nucleocytoplasmic i n t e r g r a t i o n i n Paramecium. Proceed. 9th I n t e r n ' l Congr. Genet, p a r t l y ( c a r y o l o g i a s u p p l . ) , 307-325. Sonneborn, T©M. (1954) Gene c o n t r o l l e d , aberrant n u c l e a r behaviour i n Paramecium a u r e l i a . M i c r o b i o l . Gen. B u l l .  v o l : 1 1 . 24-25.  140 Sonneborn, T.M. (1970) Methods i n Paramecium Research. In: Methods i n •'••.cell p h y s i o l o g y . D.M. P r e s c o t t (ed) v o l : 4 Academic P r e s s , New York. Sonneborn, T.M. (1975) of 14 s i b l i n g s p e c i e s .  155-178.  The Paramecium a u r e l i a complex Trans. Am. M i c r o s c . Soc. vol:94  Stevenson, I . , and F.P. Lloyd.(1971) U l t r a s t r u c t u r e of n u c l e a r . d i v i s i o n i n Paramecium a u r e l i a . I I . Amitosis of the macronucleus. Aust. J . of B i o l . S c i . vol:24,  977-987.  Suganuma, Y. (1969) E l e c t r o n microscope s t u d i e s . o n the m i t o s i s . o f b i n a r y f i s s i o n of the micronucleus of the c i l i a t e U r o s t v l a ( i n Japanese) J . Nara Med. A s s , vol:20  328-395.  .  .  Tamura, S., T. Tsuruhara and Y. Watanabe (1969). Action of n u c l e a r m i c r o t u b u l e s i n macronuclear d i v i s i o n of Tetrahymena p y r i f o r m i s . E x p t l . C e l l Res, v o l : 5 5 . 351-358. Tucker, J.B. (1967) Changes i n n u c l e a r s t r u c t u r e d u r i n g b i n a r y f i s s i o n i n the c i l i a t e Nassula. J . Cell Sci. vol:2,  481-498.  Wagner, R.C., and M.D. Rosenberg. (1973) Endocytosis i n chang l i v e r c e l l s : The r o l e o f m i c r o t u b u l e s i n vacuole o r i e n t a t i o n and movement. C y t o b i o l . v o l : 7 , 20. Walker, G.K. (1976) The f i n e s t r u c t u r e o f macronuclear d i v i s i o n i n the h y p o t r i c h c i l i a t e G a s t r o s t y l a s t e i n i i .  Protestol. vol:12(2),  271-278.  Walker, G.K., and D. Goode (1976) The r o l e of m i c r o t u b u l e s i n macronuclear d i v i s i o n i n the h y p o t r i c h c i l i a t e s G a s t r o s t y l a s t e i n i i and S t y l o n y c h i a m y t i l u s . C y t o b i o l . vol:14. 18-37. W i l l i a m s , N.E., and R.J. W i l l i a m s . (1976) Macronuclear d i v i s i o n w i t h and without m i c r o t u b u l e s i n Tetrahymena.  J . C e l l S c i . vol:20.  61-77.  Wolfe, J . (1967) S t r u c t u r a l aspects of a m i t o s i s : A l i g h t an e l e c t r o n microscope study of.the i s o l a t e d macronuclei of Paramecium a u r e l i a and Tetrahymena p y r i f o r m i s . Chromosoma v o l : 2 3 , 59-79-  141 Woodard, J . , B..Gelber and H. Swift (1961) Nucleoprotein changes during the m i t o t i c c y c l e i n Paramecium a u r e l i a . / E x p t l . C e l l Res, vol:23, 258-261. Worthington, D.H., M. Salamone and D.S. Nachtwey (1976) Nucleocytoplasmic r a t i o requirements for. the i n i t i a t i o n of DNA r e p l i c a t i o n and f i s s i o n i n Tetrahymena. Cell Tissue Kinet. v o l : 9 . 119-130. Young,.E.G., and R.B. Cambell (1947) The r e a c t i o n of b e t a - b e t a ' - d i c h l o r o d i e t h y l sulphide w i t h p r o t e i n s . Can. J . Res. v o l : 2 5 ( l ) . 37. (Sect.B. Chem. S c i . ) .  APPENDIX I  THE MODE OF ACTION OF N-methyl-N'-nitro-N-nitrosoguanidine  Nitrosoguanidine  (NTG) i s a powerful, chemical  mutagen and i s used r o u t i n e l y i n mutagen  experiments  i n v o l v i n g Paramecium (see Sonneborn, 1975). Wright NTG.  McKay and  (194-7) were the f i r s t t o s y n t h e s i z e and a n a l y s e The c y t o t o x i c , c a r c i n o g e n i c , mutagenic  chemotherapeutic interest  and  e f f e c t s have generated c o n s i d e r a b l e  i n t h i s monofunctional a l k y l a t i n g agent,.  the mutagenic  e f f e c t s of a l k y l a t i n g agents r e s u l t  t h e i r i n t e r a c t i o n on g e n e t i c m a t e r i a l , i t was that DNA was t h e most l i k e l y t a r g e t .  Since from  proposed  Young and Cambell  (1947) were amoung the f i r s t t o r e p o r t the s u s c e p t a b i l i t y of  the guanine moiety t o a t t a c k by a l k y l a t i n g  agents.  Since then, a number of authors have presented evidence t h a t the N-7  atom of guanine  Lawley and W a l l i c k (1957) of  guanine  i s the most r e a c t i v e  site.  demonstrated  t h a t the N-7 atom  i s preferentially alkylated  (support f o r t h i s  f i n d i n g came from a r t i c l e s by Reiner and Zamenhof [1957] and Alexander, et a l . ,  [1961]). 142  These r e s u l t s agree w i t h  143 the wave-mechanical s t u d i e s of Pullman.and  Pullman  (1959)  who. showed t h a t the..N-7 atom of guanine was the most n u c l e o p h i l i c s i t e i n both the u n p a i r e d base and the guanine-cytosine base p a i r .  The N-3  atom of adenine i s  also.a reactive, nucleophilic s i t e . model for. the s t r u c t u r e of DNA  The  i s also consistent with  t h i s . o b s e r v a t i o n , and shows t h a t the N-3 and the N-7  Watson-Crick  s i t e of adenine  s i t e of guanine are the most a c c e s s i b l e  of the p o s s i b l e r e a c t i v e  sites.  By u s i n g 1 4 C - l a b e l l e d  NTG  and 14  sulphonate (MMS), Lawley and Brooks  C-methyl methane-  (1963) showed t h a t  the major products from t r e a t e d , undenatured 7-methyl guanine and 3-methyl adenine. Bensted  DNA  were  S c h o e n t a l and  (1964) l a t e r showed t h a t t h i s was a l s o t r u e f o r  other a l k y l a t i n g agents.  Using l a b e l l e d m e t h y l - n i t r o s o -  urethane, S c h o e n t a l and Bensted found l a b e l l e d 7-methyl guanine and l a b e l l e d 3-methyl adenine were the major products both i n v i v o and i n v i t r o . found t h a t the N-7  S i n g e r , et a l . (1968)  atom of guanine was the primary t a r g e t  i n double-stranded (ds-) , h i g h molecular weight of DNA,  but they found t h a t t h i s was  molecules  not the case i n guanine  c o n t a i n i n g molecules of low molecular weight, such as Tobacco mosaic  v i r u s RNA  (TMV-RNA).  Under  conditions  144  f a v o r i n g the guanine r e a c t i o n ( n e u t r a l , aqueous 7-methyl guanine was t h e p r i n c i p l e - p r o d u c t . under d i f f e r e n t c o n d i t i o n s  solution)  However,  ( 6 5 % dimethylformanide)  methylated c y t o s i n e was t h e major product, and the mutagenic effect  of NTG was g r e a t l y enhanced.  They a l s o showed  t h a t t h e guanine r e a c t i o n was suppressed w i t h i n the v i r u s , but,  t h a t the c y t o s i n e  v i v o TMV-RNA,  r e a c t i o n was not,  when t r e a t e d w i t h NTG r e s u l t e d i n t h e  same r e a c t i o n product obtained in  and t h a t , i n  from p o l y c y t i d i l i c  acid  vitro.  A l k y l a t i o n o f guanine can l e a d t o s e v e r a l d i f f e r e n t types of a b e r r a t i ons. guanine, causing  i o n i z a t i o n of the guanine moiety, can  lead t o mispalring of t o c y t o s i n e  A l k y l a t i o n o f the N—7 atom o f  of the i o n i z e d guanine t o thymine  (Auerbach,  1976),  which would r e s u l t i n  a t r a n s i t i o n ffrom G-C t o A-T a f t e r DNA r e p l i c a t i o n . transitions  (purine r e p l a c i n g purine  or p y r i m i d i n e  Both  or p y r i m i d i n e  r e p l a c i n g p y r i m i d i n e ) and t r a n s v e r s i o n s pyrimidine  instead  (purine  replacing  r e p l a c i n g p u r i n e ) appear t o be  the most common e f f e c t s of NTG mutagenesis i n e u c a r y o t e s . i  Another source o f e r r o r i s the d e s t a b i l i z i n g e f f e c t of a l k y l a t i o n on the g l y c o s i d i c bond (the bond between the  sugar-phosphate backbone o f DNA and the b a s e ) .  . _ Lawley and  Brooks  \ (1963)  145 have found that at n e u t r a l  pH  there i s . a _ slow l e a c h i n g out o f the 7-alkylguanines '3-alkyladenines These gaps may  from the DNA  and  g i v i n g r i s e t o a p u r i n i c gaps.  l e a d t o t r a n s i t i o n s or t r a n s v e r s i o n s i f  f i l l e d i n c o r r e c t l y or they maye l e a d t o nonsense codons i n RNA,  a f t e r t r a n s c r i p t i o n , g i v i n g r i s e t o premature  termination  of t r a n s l a t i o n .  The  l e a c h i n g out may  lead to deletions, giving r i s e to frameshift mutations, or they may  M a i l i n g and De S e r r e s  a l t e r a t i o n s of NTG  type  l e a d t o f i s s i o n of the c h a i n  e v e n t u a l l y i n double-stranded  H.  also  and  fission.  (1970)  s t u d i e d the  genetic  mutagenesis on g e n e t i c a l l y marked  heterokaryons of Neurospora c r a s s a .  Detectable a l t e r a t i o n s  i n c l u d e d p o i n t mutations, m u l t i l o c u s chromosome d e l e t i o n s and r e c e s s i v e lethalrrnidations i n the whole genome. a n a l y s i s of NTG was  Complementation  induced ad-§B mutants showed t h a t  a h i g h frequency of G-^C  base p a i r s at the s i t e of mutation  and t h a t the major e f f e c t of NTG s u b s t i t u t i o n s from G-C  there  t o A-T.  was  i n d u c i n g base p a i r  Although G-C  to  A-T  t r a n s i t i o n s were found t o be most common i n organisms w i t h double-stranded DNA  , t h i s was  not found t o be  case i n organisms w i t h s i n g l e - s t r a n d e d DNA. Tessman (1968) found t h a t G-C i n equal p r o p o r t i o n s  t o A-T  Baker  the and  t r a n s i t i o n s occurred  i n S 13 phage, as a g a i n s t G-C  to  A-T  146 specific transitions in  Bacteriophage.  (1965)  McCalla  found t h a t NTG  was. an e f f e c t i v e b l e a c h i n g  agent,  the p r o d u c t i o n  of c h l o r o p l a s t s i n Euglena  gracilis.  The  c h l o r o p l a s t DNA  and  he  i s very r i c h i n adenine and  inhibiting  thymine,  suggests t h a t only those agents which exert  t h e i r mutagenic e f f e c t on A-T b l e a c h i n g agents.  So,  base p a i r s c o u l d be e f f e c t i v e  i n Euglena c h l o r o p l a s t DNA  it  would seem t h a t adenine i s the r e a c t i v e t a r g e t rather,than guanine.  Most of the NTG i n the DNA, DNA  mutations a r i s e as primary l e s i o n s  t h a t are converted t o f i n a l mutations at  replication.  There i s c u r r e n t  evidence t h a t NTG  act at or very near the r e p l i c a t i o n f o r k , and induced mutations a r i s e only at the  may  that  NTG  s i t e of r e p l i c a t i o n .  Auerbach (1976) suggests t h a t t h i s apparent e f f e c t i s  due  t o a g r e a t e r a c c e s s i b i l i t y of the bases t o the a l k y l a t i n g agentat r e g i o n s where t h e r e  i s strand s e p a r a t i o n .  Olmedo (1976) suggests t h a t NTG the DNA  Cerda-  a c t u a l l y i n t e r a c t s with  polymerase, causing the enzyme t o act as a pheno-  t y p i c a l l y mutagenic polymerase, which makes e r r o r s at r e p l i c a t i o n by i n c o r p o r a t i n g the wrong base.  The  possibility  of d i r e c t a c t i o n on the enzyme came from s t u d i e s t h a t showed s p e c i f i c enzymes, such as b e t a - g a l a c t o s i d a s e ,  were  1  147 very s e n s i t i v e t o treatment w i t h NTG i n a c t i v a t e d i n the presence of NTG 1976). how  and were i n f a c t  (Cerda-Olmedo,  [ I f the enzymes were i n a c t i v a t e d by NTG  could they i n c o r p o r a t e the wrong b a s e ? ] .  a l s o found that NTG inducing f a u l t y  treatment  The author  e f f e c t s p r o t e i n s y n t h e s i s by  translation.  S t u d i e s by Nestman (1975) on c o n t i n u a l l y  growing  c u l t u r e s of E s c h e r i c h i a c o l i t r e a t e d w i t h NTG  or  ( e t h y l methane s u l f o n a t e ) showed t h a t f o r NTG  mutagenesis  w i t h the mutator gene (mut H) that the mutagenic was  growth-rate dependent.  on mut  Comparison  H and w i l d - t y p e s t r a i n s  of NTG  EMS  effect  mutagenesis  (mut ), show t h a t the +  i n c r e a s e i n the r a t e o f mutation i n mut  H over mut  +  is  much g r e a t e r than c o u l d be e x p l a i n e d by an a d d i t i v e The s y n e r g i s t i c e f f e c t  of NTG  i n the r a t e of mutation.  on mut  +  i s a 48  fold  effect. increase  The s y n e r g i s t i c responses of  chemical mutagens i n t e r a c t i n g w i t h mutator genes have a p p a r e n t l y been i n t e r p r e t e d as involvement of the gene product w i t h DNA  replication  (Nestman, 1 9 7 5 ) .  The accumulating evidence (Lee and Jones, Cerda-Olmedo, e i  al.,  suggesting t h a t NTG  1968;  mutator  1973;  Schimmer and Loppes,  a c t s at the DNA  1975)  r e p l i c a t i o n fork  can be i n t e r p r e t e d i n another manner.  The  alternative  148 i s t h a t a l k y l a t e d l e s i o n s In DNA are r a p i d l y r e p a i r e d except a t the p o i n t of DNA r e p l i c a t i o n .  Thus the  only mutations t h a t become f i x e d are those o c c u r r i n g i n the, r e g i o n of the r e p l i c a t i o n f o r k .  This i n t e r p r e t a t i o n  agrees w i t h the evidence of NTG damage r e p a i r i n P. t e t r a u r e l i a  (Kimball, 1970).  t h a t when there w i t h the mutagen  Kimball  (1970) showed  i s a l o n g l a g p e r i o d between treatment (NTG) and r e p l i c a t i o n of the genome  the mutation r a t e approaches zero. damage caused by NTG  Since most of the  (transitions, transverslon, etc.)  i s r e p a i r a b l e , Kimball's  r e s u l t s suggest t h a t the  primary l e s i o n s are simply  being r e p a i r e d before  they  become f i x e d at anywhere along the DNA except at or near the r e g i o n of r e p l i c a t i o n .  APPENDIX I I  SPOT, l i n e 40-4b  A. Generation  2  time:  The mean g e n e r a t i o n time of homozygous sp_ ^ c e l l s was 5.20-.08[S.E.] hours compared t o 6.92±.27[S.E.] hours f o r the double mutant tam G/tam G;sp/sp and hours f o r w i l d type  5.00i.ll[S.E.]  cells.  B. Phenotype: C e l l d i v i s i o n was normal i n homozygous _sp c e l l s . There was no evidence of macronuclear missegregation.  I n t e r f i s s i o n c e l l s frequently  one or more l a r g e , b l a c k , c i r c u l a r ( P l a t e 13,  or m i c r o n u c l e a r  A and B ) .  cytoplasmic  contained inclusions  P o s i t i o n of these i n c l u s i o n s  varied,  but i n the m a j o r i t y of c e l l s they were a n t e r i o r l y p l a c e d . The median number of i n c l u s i o n s per c e l l was one, but o c c a s i o n a l l y up t o t h r e e i n c l u s i o n s per c e l l were observed.  High power (x 1000)  o b s e r v a t i o n s on f i x e d ,  u n s t a i n e d c e l l s , r e v e a l e d t h a t the dark i n c l u s i o n s were  149  150 composed of many c r y s t a l l i n e s p l i n t e r s enclosed i n a vesicle.  These c r y s t a l l i n e s p l i n t e r s were s i m i l a r i n  appearance t o the u r i c a c i d c r y s t a l s found through out the  the  scattered  cytoplasm of w i l d - t y p e c e l l s .  c r y s t a l s found i n the  v e s i c l e s , no u r i c  c r y s t a l s were present i n the r e s p e c t sji resembles the  Apart from  acid  c e l l ' s cytoplasm.  In  this  c_2 mutants d e s c r i b e d i n  Sonneborn (1975).  Penetrance of, the and  ranged from as low  eight-in-ten one  cells.  phenotype v a r i e d as  only one  o n e - i n - f i f t y t o as h i g h  r e v e a l e d that  i n c l u s i o n that  was  anterior  retained  fission.  inclusions  In cases where two  retained  by the  position  p r o t e r or they may  to each s i s t e r c e l l .  C.  of the  containing  i n cases where  of cases) t h i s i n c l u s i o n was  depending on the  as  Observations on i s o l a t e d c e l l s  or more i n c l u s i o n s ,  t h e r e was  between c l o n e s  by the  (majority  p r o t e r at  were p r e s e n t ,  inclusions,  both may  segregate, one  be  going  The  Genetics: The  mutation.  s_p. t r a i t i s the The  inclusions  consequence of a s i n g l e ,  recessive  disappear w i t h i n 24.00 hours  a f t e r c o n j u g a t i o n with w i l d - t y p e c e l l s .  The  phenotype  151 reappears between f i v e t o e i g h t f i s s i o n s a f t e r s e g r e g a t i o n of the h e t e r o z y g o t e s .  autogamous  T h i s gene can be used  as a marker-gene i n Paramecium matings'since the phenotype i s so d i s t i n c t i v e and easy t o observe. Conjugants after  can be i d e n t i f i e d d u r i n g p a i r i n g  s e p a r a t i o n of the conjugants.  and immediately  APPENDIX I I I THE ORIGINAL SELECTION SYSTEM  The  s e l e c t i o n system used t o r e c o v e r the v a r i a n t  l i n e s was the second c h o i c e of two p o s s i b l e The f i r s t  selection  systems.  c h o i c e , designed t o s e l e c t f o r spontaneous  MR mutants, was a more complex system, designed by Berger (per.  comm.) u s i n g a heterokaryon as the t o o l f o r  selection  (figure 20).  Homozygous pw A c e l l s were c r o s s e d t o homozygous t s l 111 c e l l s .  The conjugants were s u b j e c t e d t o heat  shock, t h i s induced r e g e n e r a t i o n of the macronuclear fragments i n conjugants. the pw A phenotype,  Exconjugant  were s e l e c t e d  clones e x h i b i t i n g  by the D r y l ' s method.  A sample o f c e l l s from each o f the pw A c l o n e s was r a p i d l y expanded and put through autogamy. genotype  was then known.  the macronucleus  The  C e l l s , homozygous f o r pw A i n  and h e t e r o z y g o u s , f o r t s l / t s l ; p w A/pw +  A  +  i n the m i c r o n u c l e i were backcrossed t o homozygous t s l 111 cells.  Again, MR was induced by heat shocking the conjugants. 152  F i g u r e 21. Formation of the pw A/pw (macronucleus)  A;tsl /tsl +  t s l / t s l ; p w A /pw +  +  A  ^micronucleus) heterokaryon. ma,  macronucleus;  mi, m i c r o n u c l e i  +  +  MUTAGENESIS  FIGURE 21  _  .  155  P h e n o t y p i c a l l y pw A exconjugant c l o n e s were s e l e c t e d f o r . The genotype  of these c l o n e s was a n a l y s e d by the same method  mentioned b e f o r e .  C e l l s homozygous f o r pw A i n the  macronucleus and e i t h e r t s l / t s l ; p w A/^pw pw A /pw A +  A  +  or  tsl /tsl; +  i n the m i c r o n u c l e i were s e l e c t e d and expanded  +  i n p r e p a r a t i o n f o r treatment w i t h NTG. has. two important v a l u e s : f i r s t l y ,  The heterokaryon  a l l the c e l l s undergoing  normal r e o r g a n i z a t i o n at autogamy, f o l l o w i n g  mutagenesis,  w i l l have m a c r o n u c l e i homozygous f o r the temperature sensitive lethals. the  These c e l l s c o u l d be k i l l e d by r a i s i n g  e n t i r e p o p u l a t i o n of mutagen t r e a t e d exautogamonts  to the r e s t r i c t i v e  temperature f o r 24.00 hours.  The  s u r v i v i n g c e l l s would be h i g h l y e n r i c h e d f o r t s l / t s l +  +  ( i n the macronucleus) which e i t h e r arose through MR or which were the progeny of c e l l s which d i d not autogamy.  undergo  Thus, p u t a t i v e , spontaneous MR mutants c o u l d  be r e c o v e r e d e a s i l y by use of the JDW column (Kung,  1971).  A number of problems were encountered i n s y n t h e s i z i n g the  heterokaryon and i n m a i n t a i n i n g the c e l l s through t o  mutagenesis. the  The major problem encountered was p r e v e n t i n g  c e l l s from going i n t o autogamy, and thereby r e v e r t i n g  t o t h e i r o r i g i n a l genotypes. new  The p r o c e s s of b u i l d i n g the  genotype took many c e l l g e n e r a t i o n s , and even i f the  c e l l s were w e l l f e d , the o l d c e l l s have a s t r o n g tendency to  e n t e r autogamy.  Normally c e l l r e j u v i n a t i o n takes p l a c e  . at  .  156  c o n j u g a t i o n , but when macronuclear r e g e n e r a t i o n i s induced,  . the c e l l s r e t a i n the p a r e n t a l macronucleus and t h e r e f o r e the  p a r e n t a l age. The f i r s t mating i s conducted between  very young c l o n e s , but many g e n e r a t i o n s a r e used up by h a v i n g t o a n a l y s e t h e genotypes a f t e r each c r o s s , and i n expanding t h e c e l l s f o r mutagenesis.  Although the  heterokayon was completed on a few o c c a s i o n s , always entered autogamy p r i o r t o mutagenesis.  cells I t was  for. t h i s reason that the s e l e c t i o n system was f i n a l l y abandoned.  Other problems encountered w i t h t h i s  system  i n c l u d e d s y n c h r o n i z a t i o n of mating p a i r s and t h e i n d u c t i o n o f MR by heat shock.  The pw A/pw A c e l l s  and t h e t s l / t s l c e l l s grow a t very d i f f e r e n t  rates  and t h e r e f o r e c u l t u r e s have t o be s e t up w i t h d i f f e r e n t amounts of food i n the c u l t u r e s .  These c u l t u r e s are then  cross-matched u n t i l mating r e a c t i v e c e l l s from both l i n e s are obtained.  A s e r i e s of minor experiments were conducted t o e s t a b l i s h the  best combination o f v a r i a b l e s i n the i n d u c t i o n o f  MR by heat shock.  Synchronized mating p a i r s were s e l e c t e d  between 5 - 5 i hours a f t e r p a i r f o r m a t i o n and r a i s e d t o t h e - r e s t r i c t i v e temperature f o r v a r y i n g l e n g t h s of time.  The  157 exconjugants were then r e i s o l a t e d and allowed t o complete two f i s s i o n s .  One daughter c e l l was k i l l e d and scored  f o r the presence of r e g e n e r a t i n g fragments. combination o f v a r i a b l e s was a temperature f o r a p e r i o d o f f i v e hours  (Table X V I I ) .  The best o f 34.5-34»7°C  Below 34.5°C  MR i n d u c t i o n was minimal and above 34.7°C was l e t h a l t o the conjugants.  The age of the conjugants was choosen t o  coincide, w i t h d i f f e r e n t i a t i o n - o f the d i v i s i o n products of the synkaryon.  158  TABLE XVII COMPARISON OF VARIABLES FOR THE INDUCTION OF MR BY HEAT SHOCK SAMPLE No.  #  TEMP °C  LENGTH SHOCK  PAIRS  # DEAD  $MR  1  50  34.3-34.5  4.00  28  20.8  2  50  it  5.00  76  -  3  50  it  6.00  27 "  4  50  II  7.00  100  5  50  34.5-34.7  4.00  40  28.0  6  50  it  5.00  27  56.5  7  50  II  6.00  20  4.7  8-  50  7.00  42  24.3  9  50  34.7-34.9  4.00  51  2.5  10  50  •t  5.00  100  -  11  50  II  6.00  100  -  12  50  m  7.00  100  —  37.2 -.  Conjugants were 5.00-5.25 hours o l d when they were r a i s e d t o the r e s t r i c t i v e temperature.  APPENDIX IV AMITOSIS  Amitosis  i s l i t e r a l l y t r a n s l a t e d as the absence o f  forming t h r e a d s . without  B a s i c a l l y , i t i s nuclear  the condensation  o f chromosomes and the f o r m a t i o n  of a t y p i c a l m i t o t i c apparatus. references  division  One of the e a r l i e s t  ( i n English) to amitosis i s that of C h i l d  (1900) when he was s t u d y i n g the r e p r o d u c t i v e  organs  i n the cestode Monieza expansa and n o t i c e d a d i s t i n c t absence o f m i t o t i c f i g u r e s i n the p r o g l o t t i d s , where the r e p r o d u c t i v e organs were d i f f e r e n t i a t i n g . confirmed  the o b s e r v a t i o n s ,  on the occurrance  He of amitotic  n u c l e a r d i v i s i o n i n the p r o g l o t t i d s , on s e v e r a l other occasions  ( C h i l d , 1902, 1904, 1907, 1910 and 1911).  Nathanson (1900) found t h a t a m i t o s i s c o u l d be induced i n S p i r o g y r a by adding a few drops of ether t o the water. T h i s i n d u c t i o n of a m i t o s i s was completely  r e v e r s i b l e by  r e t r a n s f e r r i n g j t h e S p i r o g y r a t o f r e s h water. recorded  Jordon (1913)  the e x c l u s i v e a m i t o t i c n u c l e a r d i v i s i o n i n the  epididymus of the white mouse. 159  He s t a t e d t h a t w h i l e  studying  160 spermatogenesis i n the white mouse, there was m i t o t i c f i g u r e i n evidence,  not a s i n g l e  although many n u c l e i were  i n v a r y i n g stages of a m i t o s i s .  There were many other  r e f e r e n c e s t o a m i t o s i s i n v a r i o u s t i s s u e s t h a t underwent r a p i d growth or t h a t were i n s t a r v e d c o n d i t i o n s (Holmes 1914;  Lynch, 1921,  f o r example).  More r e c e n t l y , Kocherezhkina  and Korobko (1974) observed a m i t o s i s i n the t e s t e s of. s e v e r a l s p e c i e s of B l a c k Sea crab, i n areas proliferation.  The  authors  a phenomenon i n i t ' s own  of r a p i d c e l l  suggested t h a t a m i t o s i s i s  r i g h t t h a t has a r i s e n i n response  t o an overwhelming i n c r e a s e i n the amount of n u c l e a r m a t e r i a l and  surface area.  Since the p r e r e p l i c a t i o n  macronucleus of Paramecium t e r a u r e l i a c o n t a i n s between 800-900 h a p l o i d e q u i v a l e n t s of DNA  (Woodard, et a l . ,  A l l e n and Gibson, 1972;  Berger, 1973;  i t would seem reasonable  t o agree with t h i s i d e a .  /  Morton, 1974),  1961;  161  P l a t e 1. V a r i o u s a s p e c t s of macronuclear misplacement in dividing  tam A homozygotes.  A. Normal placement and e l o n g a t i o n of the macronucleus. B. Normal placement and p a r t i a l e l o n g a t i o n of the macronucleus. C. P a r t i a l e l o n g a t i o n of the macronucleus and s l i g h t misplacement of the macronucleus toward the p r o t e r . D. R a r e l y observed misplacement of the macronucleus toward the opisthe". E. No macronuclear e l o n g a t i o n and complete r e t e n t i o n of the macronucleus i n the p r o t e r . F. P a r t i a l e l o n g a t i o n and complete r e t e n t i o n of the macronucleus i n the p r o t e r . G. Complete macronuclear m i s s e g r e g a t i o n t o the p r o t e r and a $-1 abnormal m i c r o n u c l e a r d i s t r i b u t i o n . H. Double macronucleate c e l l showing e l o n g a t i o n of one macronucleus and no e l o n g a t i o n i n the misplaced macronucleus. I . S e g r e g a t i o n without d i v i s i o n  of two m a c r o n u c l e i .  163  P l a t e 2. A comparison of complete and p a r t i a l macronuclear missegregation.  A. Complete macronuclear m i s s e g r e g a t i o n . • B, Extreme p a r t i a l macronuclear m i s s e g r e g a t i o n . C, S l i g h t  p a r t i a l macronuclear m i s s e g r e g a t i o n .  D. Equal, a m i t o t i c  division  of the macronucleus.  16$  P l a t e 3. V e g e t a t i v e tam A, and tam G homozygotes.  A. Complete m i s s e g r e g a t i o n of the u n d i v i d e d macronucleus i n tam A/tam A c e l l s . N o t i c e the s i x m i c r o n u c l e i a l i g n e d a l o n g the l i n e of f i s s i o n . B. Amicronucleate tam A/tam A  cell.  C. Amacronucleate c e l l a p p a r e n t l y mating w i t h a normal macronucleate i n d i v i d u a l , . The amacronucleate c e l l i s tam G/tam G c r o s s e d t o pw A/pw A. P..Amicronucleate d i v i d i n g c e l l undergoing macronuclear missegregation.  /  167  P l a t e 4. Improper placement of the d i v i d i n g i n tam A/tam A c e l l s ,  giving  macronucleus  r i s e t o 'fragments'  of the macronucleus at the completion of c e l l and macronuclear d i v i s i o n .  A-D.  P r o g r e s s i v e stages of c e l l d i v i s i o n showing the f o r m a t i o n of a v e g e t a t i v e macronuclear fragment (arrows).  168  P l a t e 5.  Amacronucleate v e g e t a t i v e c e l l s of tam A homozygotes showing i r r e g u l a r numbers of micronuclei.  A. Amicronucleate amacronucleate  cell,  B. Amacronucleate c e l l w i t h 1 m i c r o n u c l e u s . C. Amacronucleate c e l l w i t h 2 m i c r o n u c l e i . D. Amacronucleate c e l l w i t h 3 m i c r o n u c l e i . E. Amacronucleate c e l l w i t h 5 m i c r o n u c l e i . F. Amacronucleate c e l l w i t h 7 m i c r o n u c l e i .  V  171  Plate  6 . V e g e t a t i v e phase of the l i f e c y c l e showing i r r e g u l a r d i s t r i b u t i o n s of m i c r o n u c l e i at f i s s i o n i n tam A homozygotes.  A. The normal 2-2 d i s t r i b u t i o n of m i c r o n u c l e i . B. I r r e g u l a r d i v i d e r micronuclei.  showing a 2-0 d i s t r i b u t i o n of  C. I r r e g u l a r d i v i d e r showing a 3-3 d i s t r i b u t i o n of micronuclei. , D. I r r e g u l a r d i v i d e r showing a 4-4 micronuclei.  d i s t r i b u t i o n of  E. I r r e g u l a r d i v i d e r showing a 5 - 5 d i s t r i b u t i o n of micronuclei. F. I r r e g u l a r d i v i d e r showing a 5-1 d i s t r i b u t i o n of ' micronuclei. G and H. i r r e g u l a r d i v i d e r showing a 6 - 6 of m i c r o n u c l e i .  distribution  173  P l a t e 7. Exautogamous tam A/tam A c e l l s j u s t the f i r s t  exautogamous c e l l  completing  cycle.  A, B and C P r o g r e s s i v e stages of misplacement and f i n a l l y m i s s e g r e g a t i o n (C) of the p o s t e r i o r macron u c l e a r anlagen (ma). D and E. S i s t e r c e l l s of exautogamous tam A homozygoteshaving undergone m i s s e g r e g a t i o n of the macronuclear anlagen. N o t i c e how the m a j o r i t y of fragments passed t o the other s i s t e r c e l l . F. Normal s e g r e g a t i o n of the macronuclear anlagen at the f i r s t postautogamous c e l l d i v i s i o n .  j  175  P l a t e 8. Exautogamous tam G/tam G c e l l s j u s t completing the f i r s t postautogamous c e l l  cycle.  A. Exautogamous c e l l showing e a r l y evidence o f macronuclear r e g e n e r a t i o n i n the presence of one underdeveloped anlage. B. Normal s e g r e g a t i o n o f the macronuclear anlagen at the f i r s t exautogamous c e l l d i v i s i o n . The anlagen appear underdeveloped and fragments of the p r e z y g o t i c macronucleus are b e g i n i n g t o show signs of regeneration. C. I r r e g u l a r exautogamont w i t h 4 macronuclear anlagen. D. I r r e g u l a r exautogamont w i t h 7 macronuclear anlagen.  '177  P l a t e 9.  The completion of the f i r s t c e l l f o l l o w i n g autogamy i n tamJl/tam_A  cycle cells.  A. Normal s e g r e g a t i o n of the macronuclear anlagen. B. G r o s s l y underdeveloped macronuclear anlagen (ma) and the fragments were undergoing r e g e n e r a t i o n . C. Improper placement of one macronuclear anlage (ma), l a c k o f s u p p r e s s i o n o f DNA s y n t h e s i s i n the fragments (fgs). D. Proper alignment of r e g e n e r a t i n g macronuclear fragments ( f g s ) . E. Unequal s e g r e g a t i o n of the remaining macronuclear fragments ( f g s ) at c e l l d i v i s i o n . F. Macronuclear fragments are of g r o s s l y unequal s i z e and the d i v i d e r shows an i r r e g u l a r 4-4 d i s t r i b u t i o n of m i c r o n u c l e i (mi).  (7$  179  P l a t e 10. Exautogamous c e l l s from l i n e  21-3c.  A. Amicronucleate exautogamont showing g r o s s l y unequal development of the macronuclear anlagen. B. Amicronucleate exautogamont without macronuclear anlagen and w i t h nonregenerating p r e z y g o t i c macronuclear fragments. C. M u l t i p l e m i c r o n u c l e i i n an exautogamont without macronuclear anlagen. The p r e z y g o t i c macronuclear fragments appear s h a t t e r e d and undergoing a u t o l y s i s . D. Very e a r l y evidence of r e g e n e r a t i n g macronuclear fragments i n the p o s s i b l e absence of m i c r o n u c l e i .  P l a t e 11.  Exautogamous c e l l s b e l o n g i n g t o l i n e  21-3c.  A. An amicronucleate c e l l showing l a c k of macronuclear r e g e n e r a t i o n i n the absence of the suppressive macronuclear anlagen. N o t i c e the unusual, l a r g e food vacuole. B. The s i s t e r c e l l of the c e l l i n A. No macronuclear r e g e n e r a t i o n i s i n evidence and no food vacuoles are p r e s e n t .  183  P l a t e 12. 21-3c  conjugants showing l a c k of n u c l e a r  synchrony with the pw A/pw  A partners.  A. Prophase of the f i r s t pregamic d i v i s i o n , o n l y one micronucleus i s present i n the 2l-3c conjugant. B. Completion of the second pregamic d i v i s i o n i n the pw A/pw A conjugant. The 21-3c conjugant i s a pregamic d i v i s i o n behind. C. Amicronucleate 21-3c exautogamont p a i r i n g i n s e x u a l union w i t h pw A/pw A conjugant. D. A s i m i l a r conjugant p a i r as d e s c r i b e d f o r C , but n o t i c e the exchange of DNA a c r o s s a cytoplasmic b r i d g e .  P l a t e 13. Cytoplasmic i n c l u s i o n s  characteristic  of sp_ homozygotes.  (  

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