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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, University of 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 We accept t h i s thesis as conforming to the required standard of Zoology THE UNIVERSITY OF BRITISH COLUMBIA September, 1977 Phyllida Mary Barry Morton, 1977 In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements f o r an advanced degree at the University of B r i t i s h Columbia, I agree that the l i b r a r y s h a l l make i t f r e e l y available f o r reference and study. I further agree that permission f o r extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my department or by his representatives. It i s understood that copying or publication of t h i s thesis f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of ^oology  The University of B r i t i s h Columbia Vancouver 8, Canada V6T 1W5 Date a y W l r w r ^ 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 patterns of nuclear behaviour at c e l l d i v i s i o n and at nuclear reorganization were recovered i n separate experiments following nitrosoguanidine mutagenesis. Two 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 series of recessive genes that exert a p l e i o t r o p i c ef f e c t on macronuclear behaviour during c e l l d i v i s i o n and on the migration and attachment of trich'ocysts during trichocyst morphogenesis (Ruiz, et a l . , 1976). At binary f i s s i o n the macronuclei of homozygous tarn c e l l s f a i l to elongate and f a i l to migrate to the dorsal cortex. This leads to 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 cytokinesis, i n the l a t t e r event, the entire p r e f i s s i o n macronucleus i s retained by one s i s t e r c e l l , usually the proter. At nuclear reorganization, missegregation of the macronuclear anlagen commonly occurs either at the f i r s t or at the second post-reorganizational c e l l d i v i s i o n . Exautogamonts or exconjugants that do not receive a macro-nuclear anlage at c e l l d i v i s i o n undergo macronuclear regeneration (Sonneborn, 1940; Berger, 1973a). Improper d i s t r i b u t i o n of micronuclei occurs frequently during c e l l d i v i s i o n i n c e l l s expressing the tarn phenotype. The i i trichocysts of homozygous tarn c e l l s are abnormally shaped, are unattached and do not discharge. The t h i r d variant recovered i s unable to complete conjugation owing to abnormal-i t i e s i n the micronuclear events. TABLE OF CONTENTS ABSTRACT i i LIST OF TABLES . v i LIST OF FIGURES v i i i LIST OF PLATES x ACKNOWLEDGEMENT x i INTRODUCTION 1 MATERIALS AND METHODS 20 RESULTS 37 SECTION 1. Mutagenesis 37 SECTION I I . Genetic analysis of the variants. . 42 SECTION I I I . Phenotypic analysis 48 A. Mutations a l t e r i n g the pattern of macronuclear d i v i s i o n 4$ 1. E f f e c t s on vegetative c e l l s 48 a. Generation time 48 b. E f f e c t s on shape and size d i s t r i b u t i o n 48 c. E f f e c t s on macronuclei 51 d. E f f e c t s on micronuclei 68 e. E f f e c t on trich o c y s t s 79 2. E f f e c t s during nuclear reorganization . 82 a. Autogamy 82 b. Conjugation 86 i v V B. Variants with altered micronuclear d i s t r i b u t i o n 9 0 1. E f f e c t s on vegetative c e l l s 90 a. Generation time 90 b. Ef f e c t s on the nuclei 90 c. Penetrance and expression 92 2. E f f e c t s during nuclear reorganization. 9 4 a. Autogamy 9 4 b. Conjugation 1 0 1 DISCUSSION ., 10,5 CONCLUDING STATEMENT 1 3 0 BIBLIOGRAPHY 1 3 1 APPENDICES «• I. The mode of action of nitrosoguanidine. . 1 4 2 I I . Spot (li n e 4 0 - 4 b 2 ) 1 4 9 I I I . The o r i g i n a l s e l e c t i o n system 1 5 2 IV. Amitosis 1 5 9 PLATES 1 6 1 LIST OF TABLES Table. Page I. Mutagenesis experiments: A comparison of the frequency of exautogamous death with the y i e l d , , 40 I I . Variant l i n e s recovered 41 I I I . Exautogamous segregation of the tarn genes • . 43 IV. Results of mating i n the variant 21-3c , . 44 V. Complementation test between tarn genes . . 46 VI. Frequency of complete macronuclear missegregation i n the tarn A/tam A;am/am double mutant 47 VII. A comparison of the frequency of p a r t i a l and complete macronuclear missegregation i n tarn A and tarn G 61 VIII. The effect of temperature on nuclear missegregation i n homozygous tarn A and tarn G c e l l s . . . . . . . 67 IX. Comparison of the number of micronuclei between homozygous tarn A and tarn G and wild-type c e l l s 69 X. Macronuclear segregation at c e l l d i v i s i o n . 72 XI. The frequency of missegregation of anlagen at the f i r s t and second exautogamous c e l l d i v i s i o n . 87 XII. Variation i n the number of micronuclei i n morphostatic c e l l s of l i n e s 10-la and 19-2b 91 XIII. Micronuclear segregation at c e l l d i v i s i o n . 93 XIV. Number of micronuclei at d i v i s i o n i n v i \ v i i 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 at 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 98 XV I . V a r i a t i o n i n the number of n u c l e i i n exautogamoussf i rs t c e l l c y c l e d i v i d e r s . . . 99 X V I I . Comparison of v a r i a b l e s f o r the i n d u c t i o n of MR by heat shock 158 \ LIST OF FIGURES Figure Page 1. Section through the cortex of Paramecium  I5etraurelia ' • • 3 2. Internal morphology of P_. t e t r a u r e l i a . . . . 7 3. Nuclear events during p r e f i s s i o n morphogenesis 10 4. Nuclear events during the sexual process . . 14 5. Nuclear events during autogamy 17 6. I s o l a t i o n scheme of the F-^progeny i n a cross involving a tarn gene 32 7. Probit analysis_of tarn A c e l l cycle lengths . 49 8. A comparison of c e l l morphology 52 9. Missegregation of the macronucleus at f i s s i o n i n c e l l s expressing the tarn phenotype 54 10. A comparison of macronuclear p r e f i s s i o n morphogenesis . 57 11. Penetrance of the tarn A and am genes as a function of c l o n a l age 62 12. The frequency of complete macronuclear missegregation i n tarn A/tam A and am/am c e l l s as a function of clonal age 64 13. Diagram of the experiment comparing the number of micronuclei i n related pairs of s i s t e r c e l l s 70 14. Theoretical diagram deomonstrating how large numbers of micronuclei may a r i s e i n c e l l s expressing the tarn phenotype . . . . 73 v i i i i x Figure page 1$. A comparison of early, middle and l a t e dividers 75 16. I l l u s t r a t i o n of the difference i n micronuclear placement i n ±/± and tarn A/tam A di v i d i n g c e l l s 77 17. The difference i n trichocyst morphogenesis 80 18. The experiment comparing the frequency of macronuclear missegregation at the f i r s t and second exautogamous d i v i s i o n . . . . $4 19. The effect of age on the frequency of micronuclear missegregation 95 20. Abnormal mating configurations from +/+ X 21-3c 103 21. Formation of the pw A/pw A;tsl^tsl +(mac.) tsl/tsl+/pw A+/pwAA+(mic.) heterokaryon . . . . 153 LIST OF PLATES Plate page " 1. Various aspects of macronuclear mis-placement i n di v i d i n g tarn A homozygotes. . . . 161 2. A comparison of complete and p a r t i a l macronuclear missegregation 163 3. Vegetative tam A and tarn G homozygotes . . . . 165 4. Improper placement of the div i d i n g macro-nucleus i n tam A/tam A c e l l s , giving 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 . 167 5. Amacronucleate vegetative c e l l s of tam A homozygotes showing i r r e g u l a r numbers of micronuclei 169 6 o 6. Vegetative phase of the l i f e cycle showing i r r e g u l a r d i s t r i b u t i o n of micronuclei at f i s s i o n i n tam A homozygotes 171 7. Exautogamous tam A/tam A c e l l s just complete ing the f i r s t postautogamous c e l l cycle . . . 173 8. Exautogamous tam G/tam G c e l l s just complet-ing the f i r s t postautogamous c e l l cycle . . . 175 9. The completion of the f i r s t c e l l cycle following 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 181 12. 21-3c conjugants showing lack of nuclear synchrony with pw A/pw A partners 183 13. Cytoplasmic inclusions c h a r a c t e r i s t i c of sp homozygotes. . . . . . . . 185 x ACKNOWLEDGMENT The author wishes to thank Dr. J.D. Berger f o r his supervision of t h i s study and he l p f u l c r i t i s m of the following manuscript. Special thanks go to my husband, Glenn Morton, f o r h i s help with the computing and the photography, f o r his understanding and encouragement, and f o r his u n f a i l i n g moral and f i n a n c i a l support. Thanks are also warmly extended to Carol Pollack and Don Jones f o r t h e i r many i n s p i r i n g discussions at seminars (and wholenars) and i n the laboratory, and for t h e i r very spe c i a l friendship. - This research was supported by N.R.C. operating grant A67-6300 to Dr. J.D. Berger. x i INTRODUCTION The i n i t i a l aim of t h i s study was to analyse the reversible i n t e r a c t i o n between the postzygotic macro-nucleus and the prezygotic macronuclear fragments i n Paramecium t e t r a u r e l i a (Sonneborn, 1975) exconjugants. This i n t e r a c t i o n r e s u l t s i n selective suppression of DNA synthesis i n macronuclear fragments when a new, immature macronucleus i s present (Berger, 1973a). It was hoped that the nature of the i n t e r a c t i o n between fragments and anlagen could be elucidated through the selec-t i o n and analysis of induced mutations which alt e r e d the normal suppressive e f f e c t of macronuclear anlagen on DNA synthesis i n macronuclear fragments. It was expected that i n such mutant c e l l s macronuclear regeneration would occur i n the presence of macronuclear anlagen;- The selection system was successful and three putative mutants which underwent macronuclear regeneration were recovered 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 variants (tarn A and tarn G) revealed that the cause of macronuclear regeneration i n exautogamont c e l l s was due not to 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 prezygotic macronuclear 2 fragments but to a l t e r a t i o n s i n nuclear behaviour at c e l l d i v i s i o n , which stem from improper cytoplasmic l o c a l i z a t i o n of the p r e d i v i s i o n n u c l e i . Analysis of the t h i r d variant revealed abnormal behaviour of the micronuclei during the sexual process. In view of the findings, t h i s study focused upon the analysis of the three variants recovered following n i t r o -soguanidine mutagenesis. It i s hoped that a further under-standing of nuclear morphogenesis at c e l l d i v i s i o n and at nuclear reorganization w i l l be gained from the r e s u l t s obtained. Before continuing with the analysis some back-ground information about the organism and i t ' s l i f e cycle w i l l be presented. C e l l Cortex The holotrichous 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 0.6V ; (Kaneda and Hanson, 1976). The c e l l cortex contains longitudinal rows of c o r t i c a l units known as k i n e t i e s . Each unit consists of one or two c i l i a and basal bodies (kinetosomes), a perisomal sac and a long filament (kinetodesma) that runs p a r a l l e l to the kinety from the side of the posterior basal body (Figure 1). The (1) In t h i s thesis a l l c e l l cycle stages are indicated by decimal fract 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 cycle. Figure 1. Section through the cortex of P. t e t r a u r e l i a . (After Ehret an'd Powers, 1959) / / 4 FIGURE 1 5 anterior and posterior boundaries of each c o r t i c a l unit are defined by t r i c h o c y s t s , one at each boundary. The trichocysts are ejectosomes between two and f i v e micrometers i n length, aligned just beneath the c e l l ' s surface and perpendicular to the plasma membrane. They are surrounded by a single membrane and are composed of three parts: the body, the t i p and the sheath. Both the body and the t i p of the trichocyst are composed of a t r y p s i n - d i g e s t i b l e protein and. the sheath i s composed of a pepsin-digestible protein and glycoprotein (Esteve, 1974) . In the mature tri c h o c y s t , the protein i s arranged i n a c r y s t a l l i n e struc-ture with a p e r i o d i c i t y of 7 - l6nm (Pollack, 1974) . The structure of t r i c h o c y s t s changes dramatically upon extrusion. According to Kaneda and Hanson (1974) the c e l l surface can be divided into two zones: the adoa?al zone (which i n -cludes the upper l e f t quadrant, the o r a l groove and the vestibule) related almost e n t i r e l y to feeding, and the anoral zone (which includes the remaining c e l l surface) concerned with m o t i l i t y . The cytoproct (anus) i s located on the'ven-t r a l (oral) surface i n the posterior part of the c e l l . Two c o n t r a c t i l e vacuole pores are found on the dorsal (aboral) surface l y i n g i n the interkinety spaces, one anterior and the other posterior. Each of these vacuoles i s about one t h i r d of the distance from one end of the c e l l . 6 Nuclei Paramecium t e t r a u r e l i a possess two types of n u c l e i . The wild type c e l l contains two, small, round, d i p l o i d , germinal micronuclei and one larger, polygenomic, somatic macronucleus (Figure 2 ) . During vegetative growth the macronucleus i s responsible f o r the biosynthetic events which direct c e l l phenotype and c e l l function. Macronuclear RNA synthesis and output i s considerable and masks the small amount of RNA synthesized by the micronuclei (Pasternak, 1 9 6 7 ) . . During the morphostatic portion of the c e l l cycle ( 0 . 1 - 0 . 7 , i n t e r f i s s i o n age, Kaneda and Hanson, 1974) the macronucleus i s round, and l i e s adjacent to the dorsal side of the buccal cavity. S t r u c t u r a l l y , the macronucleus i s complex. It contains many small, electron-dense chromatin bodies and numerous, larger n u c l e o l i (Jurand, Beale and Young, 1962; Dippell and Sinton, 1963; Stevenson and Lloyd, 1971) . The small bodies have a DNA equivalent of about one - chromosome and they are interconnected by very f i n e DNA threads (Wolfe, 1967 i n Paramecium t e t r a u r e l i a and Tetrahymena pyriformis; Morat, 1973 i n Colpidium campylum). The DNA content of a macronucleus i s approximately equivalent to 860 haploid genomes (Woodard, et a l . , 1961; A l l e n and Gibson, 1972; Berger, 1973b; Morton, 1 9 7 4 ) . Macronuclear S phase has been reported to begin at 0.25 of the c e l l cycle, and continues up to karyokinesis (Kimball, et a l . , I960; Woodard, et a l . , 1961; Berger, 1971) . There i s no G 9 period i n P_. t e t r a u r e l i a . FIGURE 2. Internal morphology of P. t e t r a u r e l i a . c i , c i l i a ; cv, c o n t r a c t i l e vacuole; ma, macronucleus; mi, micronuclei; og, oral groove; f v , food vacuole; t r , t r i c h o c y s t s . 8 Interpretations of the organization of the macronucleus i n members of the Oligohymenophora are varied. There have been several conjectures ranging from random r e p l i c a t i o n and assortment of unordered chromosomes to the macronucleus being made up of complete genome, d i p l o i d subunits (see Nyberg, 1976). Probably the most accepted version of macronuclear organization i s that the macronucleus i s made up of haploid subunits containing complete genomes. This at least appears to be true i n Tetrahymena (see Orias and Flacks, 1975). Division of the, macronucleus i s amitotic, that i s , without condensation of chromosomes or the formation of 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 cation on the dorsal side of the gull e t and moves to the centre part of the c e l l . (Kaneda and Hanson, 1974). In t h i s p o s i t i o n the' macronucleus begins to swell but maintains i t s spherical, shape. At about i n t e r f i s s i o n age 0.85 the macronucleus begins to elongate very s l i g h t l y and migrates towards a subcortical location on the dorsal aspect, of the c e l l (Figure 3). Simult-aneously, microtubules appear i n the cytoplasm p a r a l l e l to the nuclear envelope but without connection to i t (Stevenson and Lloyd, 1971). Repositioned i n the subcortical region, the macronucleus elongates and, at the same time, the extra-nuclear microtubules grow along the longitudinal axis of the Figure 3 . Nuclear events during ppa?edivision morphogenesis., fv, food vacuoles; ma, macronucleus; mi, micronuclei. 0 . 7 - 1 . 0 , i n t e r f i s s i o n age of the c e l l . At 0 .7 the macronucleus moves away from the dorsal side of the buccal cavity and assumes a new location i n the centre of the c e l l . At age 0 . 8 the macronucleus begins elongation and repositions i n a subcortical location on the dorsal side of the c e l l . At age 0 .85 the micronuclei have completed mitosis and the macronucleus has elongated. Con-s t r i c t i o n of the macronucleus occurs at about age 0 . 9 and c e l l d i v i s i o n i s completed at age 1 . 0 . m a 0.7 0.8 0-85 0.9 FIGURE 3 12 c e l l . When elongation commences, intranuclear microtubules 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 dorsal cortex of the c e l l . Simultaneously, the intranuclear micro- -tubules a l i g n along the longitudinal axis of the nucleus and the extranuclear microtubules become reduced i n number. During.constriction of the macronucleus, a large number of microtubules appear i n p a r a l l e l arrangements along the long-i t u d i n a l axis of the d i v i d i n g macronucleus i n the isthmus region. Karyokinesis and cytokinesis occur concurrently. Upon completion of f i s s i o n the daughter macronuclei contract into the t y p i c a l oval form of the i n t e r f i s s i o n c e l l and migrate back to the i n t e r f i s s i o n p o s i t i o n at the dorsal side of the g u l l e t . Recently, Inaba and Kudo (1972) found that i n P. multimicronucleatum the extranuclear microtubules, which arrange just p r i o r to c e l l d i v i s i o n , do connect with pores of the macronuclear and micronuclear envelopes. At the completion of macronuclear d i v i s i o n no trace of the i n t r a -nuclear microtubules remains. It i s not clear what role the i n t a - and extranuclear microtubules play during karyokinesis. It might be suggested that the intranuclear microtubules may pa r t i c i p a t e i n chromosome movement during nuclear d i v i s i o n . However, attachments between these microtubules and chromatin bodies have not been observed. It i s generally assumed that the microtubules are involved i n nuclear elongation (e.g. Inaba and Kudo, 1972) . 13 The micronuclei l i e i n small depressions on the surface of the macronucleus. They are small, round nuclei surrounded by a nuclear envelope that remains intact during mitosis. The micronuclear S phase begins about 50^ of the way through the c e l l cycle and l a s t s f o r about 80 minutes (Pasternak, 1 9 6 7 ) . D i v i s i o n of the micronuclei occurs at about i n t e r f i s s i o n age 0.85 and i s characterized by an intranuclear mitotic spindle (Stevenson and Lloyd, 1 9 7 1 ) . The L i f e Cycle of Paramecium t e t r a u r e l i a The mean c e l l cycle length under normal culture con-diti o n s (27°C and well fed) i s approximately 5.00 hours f o r wild type c e l l s . The vegetative part of the l i f e cycle may l a s t f o r several hundred c e l l generations. The c e l l s event-u a l l y age and die unless meiosis, f e r t i l i z a t i o n and the form-ation of a new macronucleus (nuclear reorganization) takes place. Three types of nuclear reorganization occur i n P.  t e t r a u r e l i a : 1. Conjugation (Figure 4 ) C e l l s of opposite mating type (Sonneborn, 1 9 3 7 ) form couplets by j o i n i n g at t h e i r oral surface. The micronuclei undergo meiosis and give r i s e to eight haploid products. Seven of these degenerate and one migrates to the paroral cone (a unique structure which forms near the mouth during Figure 4. Nuclear events during the sexual process (conjugation) i n P. t e t r a u r e l i a . fv, food vacuoles; ma, macronucleus; mi, micronuclei. a. micronuclei i n early prophase of the f i r s t pregamic d i v i s i o n ; b. micronuclei i n the t y p i c a l crescent stage of the f i r s t pregamic d i v i s i o n ; c. elongation and early fragmentation i n the macronucleus, completion of the f i r s t pregamic d i v i s i o n ; d. skein formation i n the macronucleus, completion of the second pregamic d i v i s i o n ; e. macronuclear fragmentation, formation of the isogamic nuclei (migratory and stationary); f . r e c i p r o c a l exchange of the male pronucleus; g. formation of the zygotic nucleus (synkaryon); h. completion of the second mitotic d i v i s i o n of the synkaryon; i . uncoupling of the exconjugants, d i f f e r e n t i a t i o n of the synkaryon products; j . segregation 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 meiosis) where i t completes a mitotic d i v i s i o n to give r i s e to one stationary and one migratory isogametic pronuclei. Reciprocal exchange of the migratory pronuclei takes place between the c e l l s . Fusion of the exchanged nuclei with the indigenous stationary nuclei occurs to form d i p l o i d , zygotic nuclei (synkaryon) i n both c e l l s . The synkaryon divides twice, m i t o t i c a l l y . In each c e l l two products become macro-nuclear anlagen and two become the new micronuclei. The vegetative nuclear complement i s restored at the f i r s t c e l l d i v i s i o n when the anlagen are segregated to the daughter c e l l s . During conjugation, the macronucleus breaks down into fragments (about 35/cell). These fragments are randomly segregated to daughter c e l l s when the vegetative cycle i s resumed. DNA synthesis i s suppressed i n the fragments and eventually they are autolysed (Berger, 1973a). 2. Autogamy (Figure 5) Autogamy takes place i n uncoupled c e l l s . It i s a form of ' s e l f i n g ' . Micronuclear events proceed as described f o r conjugation, but the isogametic pronuclei fuse immediately a f t e r t h e i r formation, foregoing any nuclear exchange with another c e l l . Since the pronuclei which fuse to form the synkaryon i n an autogamous c e l l are s i s t e r mitotic products of a single haploid nucleus, the synkaryon i s completely homozygous. Figure 5. Nuclear events during autogamy i n P. t e t r a u r e l i a . f v , food vacuole; ma, macronucleus; mi, micronucleus. a. micronuclei .in early prophase of the f i r s t pregamic d i v i s i o n ; b. crescent stage of the f i r s t pregamic d i v i s i o n ; c. completion of the, f i r s t pregamic d i v i s i o n ; d. skein formation of the macronucleus, completion of the second pregamic d i v i s i o n of the micronuclei; e. fragmentation of the macronucleus, formation of the isogametic pronuclei; f . fusion of the pronuclei to form the synkaryon; g. and h. f i r s t and second mitotic d i v i s i o n of 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. segregation of the macronuclear anlagen at the f i r s t c e l l d i v i s i o n ; 1. vegetative cycle resumed. FIGURE 5 19 3. Cytogamy Cytogamy does not appear to be a phenomenon i n i t s own ri g h t , but i s , rather, a f a i l u r e of the c e l l s to complete conjugation. Cytogamy can only take place during conjugation, since i t i s essentually 'autogamy at conjugation'. Coupled c e l l s follow the same nuclear events already described f o r conjugation with the exception that exchange of the migratory pronuclei does'not take place. Cel l s that complete t h i s 'false conjugation' are completely homozygous. 20 MATERIALS AND METHODS Culturing Techniques C e l l s were grown either i n cerophyll or i n grass media (pH6.8 - 7.0) with Aerobacter aerogenes as the food organism (Sonneborn, 1970). Stock cultures were kept at 17°C and fed every two weeks. A l l experiments were conducted 'at room temperature unless otherwise s p e c i f i e d . 12.5 gms 3.75 gms 5,000 mis The cerophyll (grass) and buffer were added to f i v e l i t e r s of d i s t i l l e d water and boiled f o r f i v e minutes. The culture f l u i d was, then bottled and autoclaved. The culture f l u i d was inoculated with the food organism 21+ hours before use.. The pH was adjusted immediately before use. Stocks — — — — • — ~ • \ The various strains of Paramecium t e t r a u r e l i a used i n t h i s study are described i n Table 1. Culture Media cerophyll or grass powder Na2HP0^ H 20 ( d i s t i l l e d ) TABLE I 21 STOCKS USED IN THE COURSE OF THIS STUDY STOCK GENE REMARKS AND REFERENCES 51.S Wild-type stock (Sonneborn, 1975) d4-43 am, nd6 Both recessive mutations were recovered a f t e r UV mutagenesis (Sonneborn, 1954) and characterized by N o b i l i (1959, 1961). D4-43 i s derived from stock d4 -2 . The am gene causes missegregation of the macronucleus at c e l l d i v i s i o n and mis-segregation of anlagen at the f i r s t and/ or second exconjugant (or exautogamont) c e l l d i v i s i o n . The am gene also causes missegregation of micronuclei at c e l l d i v i s i o n . The nd6 gene, when homozygous, causes f a i l u r e of tric h o c y s t s to d i s -charge when the c e l l i s stimulated with an i r r i t a n t ( p i c r i c a c i d ) . d 4 - l l 6 ftA This recessive mutation was recovered a f t e r X-ray treatment (Pollack, 1970). The mutation causes incomplete morpho-genesis of t r i c h o c y s t s . The tric h o c y s t s are unattached to the c e l l ' s cortex, are t i p l e s s , are f o o t b a l l shaped, and do not discharge (Pollack, 1970). d4-103 f t B This recessive mutation was recovered a f t e r treatment of 51.S c e l l s with NTG. ' The phenotype i s s i m i l a r to that of ftA but i s not a l l e l i c with ftA (Pollack7~T970). d4-94 pwA This recessive mutation was recovered a f t e r NTG mutagenesis of stock 51.S c e l l s . Homozygous pwA cells-do not avoid i n solutions higE i n Na+ K or Ba*(Kung, 1971). d4-106 stA This recessive mutation was recovered a f t e r NTG treatment of stock 51.S c e l l s . The gene was characterized by Pollack (1970, 1974). When homozygous i t causes incomplete morphogenesis of t r i c h o c y s t s . Trichocysts are stubby i n shape and are usually tipl:Css3s or have skewed t i p s . Some trichocysts are attached to the c e l l ' s cortex and can discharge. Most of them are unattached. 22 STOCK GENE REMARKS AND REFERENCES d4-107 stB d4-1030 tamA d4-1031 tamG d4-108 tam8 tam38 t - 3 3 This_recessive,mutation i s s i m i l a r i n phenotype to stA with the exception that stB does not show/a wild-type phenotype under mild starvation as stA does. This recessive 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 nuclei at c e l l d i v i s i o n . It also causes improper trichocyst morphogenesis. The t r i c h o -cysts are f o o t b a l l shaped, t i p l e s s and unattached to the cortex. They do not discharge when stimulated with p i c r i c acid, This recessive mutation was recovered a f t e r NTG mutagenesis of stock 5 1 .s c e l l s . Tam G has a similar phenotype to tam A. In addition to the eff e c t on nuclear mis-segregation and trichocyst morphogenesis, tam G c e l l s frequently have extra nuclei at nuclear reorganization. Trichocysts are stubby. This recessive mutation was recovered from NTG treated stock 5 1 .s c e l l s (Beisson and Rossignol, 1975) . The mutation causes missegregation of nuclei at c e l l .division and f a i l u r e of trich o c y s t s to discharge. The t r i c h o c y s t s i n tam/tam c e l l s are not aligned and are unattached (Sonneborn, 1974; Beisson and Rossignol, 1975 and Ruiz et a l . , 1976) . This recessive mutation was recovered from UV treated stock d4 -2 c e l l s (Adoutte and Beisson, 1970) . When homozygous, tam 38 causes nuclear missegregation at c e l l d i v i s i o n ; f o o t b a l l shaped, nondis-charge tri c h o c y s t s and extra nuclei at nuclear reorganization. Tam 38 i s very s i m i l a r to tam Gj (except f o r the shape of the trichocysts) and tam A. This recessive mutation also causes the tam phenotype when homozygous. T -33 was recovered from NTG treated stock d4 -2 c e l l s . STOCK GENE REMARKS AND REFERENCES 23 d.4-108 tam21 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 with NTG (Beisson and Rossignol, 1975). Trichocysts are foot-b a l l shaped, unattached and f a i l to d i s -charge when the c e l l i s stimulated with p i c r i c a c i d . The growth r a t e ' i s slow (2-3 f i s s i o n s per day) and the c e l l s die within 36 hours of being raised to the r e s t r i c t i v e temperature (35°C). d4-85 t s l l l This i s a temperature-sensitive, recessive, l e t h a l mutation. It was recovered a f t e r treatment of d4-2 c e l l s with UV. T s l l l /  t s l l l w i l l die within 48 hours a f t e r being raised to the r e s t r i c t i v e temperature (Beisson and Rossignol, 1969). 24 Mutagenesis Populations of stock 5 1 . s c e l l s (wild type) were expanded to about one m i l l i o n c e l l s . The c e l l s were then concentrated by mild centrifugation ( 1 . 0 min at 1 0 0 0 rpm) and resuspended i n 1 0 0 mis of Dryl's solution (Dryl 1 9 5 9 ) . The chemical mut-agen N-methyl-N'-nitro-N-nitrosoguanidine (NTG,CH^.N(N0).C(NH). NH.NOg, Sigma chemicals) was dissolved i n 1 0 0 mis of Dryl's solution to a concentration of 75 ug/ml (Kung 1 9 7 1 , Peterson, 1 9 7 5 ) . The concentrated c e l l suspension was mixed with the dissolved NTG and allowed to stand f o r 50 to 60 minutes. After treatment the c e l l s were washed three times i n s t e r i l e Dryl's solution and suspended i n fresh culture f l u i d with enough bacteria to allow f o r three to four doublings of the c e l l population. This culture was maintained f o r 24 to 4 8 hours to allow f o r the exhaustion of the food and the occur-rence of autogamy. Precautions Dilute n i t r i c a c i d was available at a l l times during mutagenesis. A l (Contaminated u t e n s i l s and glassware were placed i n d i l u t e n i t r i c a c i d containers where they were l e f t f o r one week i n the l i g h t . Mutagenesis was conducted i n a plexiglass safebox i n a fumehood. 25 Population"Counts Population counts were made by s e r i a l d i l u t i o n of 1.0 ml of the c e l l culture. The average was determined from direct counts of three or more samples. Test f o r Autogamy 1 ' Between 30-50 c e l l s were placed i n a drop of Dryl's solution on a clean microscope s l i d e . A drop of Dippell sta i n (Dippell, 1955) was added to the drop of c e l l s and covered with a cover s l i p . Observations were made using a low power (20x) objective. The cytoplasm stained green i n contrast to the nuclei which stained dark brown. C e l l s i n autogamy contained many macronuclear fragments rather than the compact nucleus seen i n vegetative c e l l s . The lowest l e v e l that was acceptable was 95$ autogamy. Determination of Vegetative 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 treated c e l l population and from an untreated control population, were made and allowed to grow f o r two days. i The frequency of vegetative c e l l death was ascertained. Clones which contained no more than four c e l l s were scored as dead (normal l i n e s had several hundred c e l l s ) . The frequency of vegetative death gave an in 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 subtracted from the per-, 26 centage of exautogamous death. The percentage of death i n the.control i s o l a t e s indicated the l e v e l of t o x i c i t y of.Dryl's solution. Dryl's solution i s a physiological s a l t solution which can be toxic i f the c e l l s are l e f t i n i t too long. Determination of Exautogamous Death Following 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 to grow f o r two. to three days. Clones with four c e l l s or fewer were scored as dead. Since autogamy r e s u l t s i n complete homozygosis of the genome, the frequency of exautogamous death gives an i n d i c -ation of the r e l a t i v e frequency of l e t h a l mutations produced by the mutagen and i s thus an in d i c a t i o n of the effectiveness of the mutagenic treatment. Mutagenic experiments with an exautogamous death l e v e l of below 45% were considered unsuc-cessful and therefore discarded. Method of Selection Mutagen treated exautogamous c e l l s were placed i n p e t r i dish cultures and fed. Early dividers were selected from the " p e t r i dish cultures and were i s o l a t e d into depression s l i d e wells. S i s t e r c e l l s were allowed to 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 dried on albumin-coated s l i d e s i n rows. The s l i d e s were stained with Azure A (Berger, 1969), and the c e l l s were scored f o r the presence of regener-27 ating macronuclear fragments and f o r abnormal numbers of micronuclei and macronuclear anlagen. The l i v i n g s i s t e r c e l l s of stained c e l l s with abnormal nuclear complements were retained as putative mutants. Test f o r Trichocyst Discharge A small sample of l i v e c e l l s was placed i n a drop of Dryl's solution on a microscope s l i d e . A drop of saturated p i c r i c acid was added. Wild-type c e l l s discharge t h e i r entire complement of trich o c y s t s within a second. C e l l s homozygous f o r tarn, jst, f t and nd f a i l to discharge t r i c h o -cysts. Discharged trichocysts appear as a halo of t i n y hairs when viewed with a low power objective. Test f o r the Presence of Trichocysts One or two c e l l s were placed i n a minute drop of Dryl's solution 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 to rupture the c e l l s . The c e l l s were examined with a negative phase con-tra s t o i l immersion objective. The trichocysts could be seen as l i g h t spindle shaped elements on a dark background. Analysis of Phenotypic Age Dependence Ce 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 at 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 , fed and allowed to complete one d i v i s i o n . One s i s t e r c e l l from each i s o l a t e was tested f o r autogamy. The l i v i n g s i s t e r c e l l was cloned. Each day a small, newly divided c e l l was transferred to a fresh depression s l i d e and the number of d i v i s i o n s completed by the c e l l s i n the old depression slidevwere recorded. Every three days, a f t e r the transfer of a young c e l l , the three previous depression s l i d e clones were pooled and trans-ferred to a p e t r i dish culture. On the following day 100 dividers were selected and dried i n rows on s l i d e s . This procedure was followed so that at approximately every 10 f i s s i o n s a sample of 100 dividers f o r each l i n e was taken u n t i l the l i n e s were 60-70 f i s s i o n s old or u n t i l they went into autogamy. Test f o r Mating Reactivity C e l l s were grown i n tube cultures with excess food f o r several days. After f i v e days the cultures were allowed to exhaust the medium. After 30-36 hours the c e l l s were concen-trated by mild centrifugation and the c e l l p e l l e t was placed i n a drop of Dryl's solution i n the well of a depression s l i d e . C e l l s that were mating reactive would clump i n the bottom of the well. Non-mating reactive cells 1"would disperse. 29 Synchronizing Mating Pairs Mating reactive cultures of opposite mating type were mixed and loose mating pairs were allowed to form. The mating culture was l e f t f o r two hours a f t e r the formation of the f i r s t l a s t i n g c e l l contacts. Couplets were then drawn into a large bore micropipette and expelled f o r c e f u l l y . I f the pa i r remained t i g h t i t was considered to be two hours old (Berger 1969). A l l pa i r s that came, apart or appeared loose a f t e r expulsion from the pipette were discarded. The start of conjugation was taken as the point at which c e l l s established l a s t i n g contact. Induction of Macronuclear Regeneration Synchronous mating pairs were shi f t e d to a water bath at 34.5°C at 5-g- h o u r s a f t e r the start of conjugation. The pairs were kept at the high temperature f o r f i v e hours and were then returned to room temperature. This procedure resultswin macronuclear regeneration i n a small percentage of the exconjugants. Selection of Ce 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 the use of suitable genetic markers i n the parental macro-n u c l e i . The marker, pwA (Rung, 1971; Chang et a l . , 1974) (2) This i s the time when the d i f f e r e n t i a t i n g synkaryon products are most vulnerable to shock treatment. .30 was used i n one parental macronucleus and a mutation f o r trichocyst nondischarge (nd6, tam A or tam G)was used i n the other. Homozygous pwA c e l l s f a i l to show the t y p i c a l avoiding reaction of wild-type c e l l s . Exautogamont c e l l l i n e s undergoing M.R. 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 depression s l i d e cultures and allowed to complete two f i s s i o n s . Two of the four daughter c e l l s were dried on s l i d e s , stained and scored fo r the presence of regenerating macronuclear fragments. The Problem of Phenomic Lag1 When the f i l i a l and parental genotypes d i f f e r , a cer t a i n amount of time i s required before the phenotype corresponding to the f i l i a l genotype comes into expression. This i s be-cause exconjugants r e t a i n the cytoplasm of t h e i r parents. This persistence of the parental phenotype into the F-^  gen-eration i s known as phenotypic or phenomic lag (Sonneborn, 1953; Berger, 1976) and i s analogous to the occurrence of maternal e f f e c t s i n higher organisms. Thus, when homozygous tam A c e l l s are crossed to wild-type c e l l s , some of the descendants of the tam A/tam A parents w i l l show macronuclear missegregation at the f i r s t or second d i v i s i o n of the excon-jugant. Missegregation i n tam,gives r i s e to c e l l l i n e s which have no macronuclei and which consequently undergo macro-nuclear regeneration. The expression•of the parental pheno-31 type i n these M.R. l i n e s can lead to errors i n determination of phenotypic r a t i o s . To overcome the problem of phenomic lag, s i s t e r c e l l s produced at each of the f i r s t f i v e f i s s i o n s of the excon-jugants were i s o l a t e d and t h e i r phenotypes were determined (Figure 6 ) . Lines showing the parental (tarn) phenotype were eliminated. Cytological Techniques 1. Fix a t i v e : a) absolute ethanol 3 parts b) g l a c i a l acetic acid 1 part f i x f o r 20 min. 2 . Whole c e l l mounts: A t h i n layer of Meyer's egg albumin was spread on a clean microscope s l i d e with the bevelled edge of a second s l i d e . The s l i d e was heated gently over a bunsen burner u n t i l the albumin steamed but was not scorched. Ce l l s were then placed i n microdrops, aligned i n rows on the prepared s l i d e , and allowed to dry f l a t on the s l i d e . ' The s l i d e s were then f i x e d and stained. 3. Stains and staining procedures: a) Azure A Figure 6. Iso l a t i o n scheme of the F-^  progeny i n a cross involving a tarn gene. ma, macronucleus; mi, micronuclei; fg , fragments. Isolates showing the parental (tarn) phenotype were discarded. 34 10 mis of 0 .5% Azure A 1 .5 mis of 1 N HC1 0.15 gms of Sodium Metabisulphite Procedure: i ) dry c e l l s on albuminized s l i d e s i i ) f i x f o r 20 min. i i i ) hydrolyse f o r 11 min. i n 1 N HC1 at 60°G iv) s t a i n f o r 15 min. v) rinse with U^O vi) counter stain v i i ) rinse i n E^O ( d i s t i l l e d ) v i i i ) a i r dry ix) mount b) Dippell st a i n Combine 10 .5 parts of acetoamine with 4 .5 parts of 45% Acetic a c i d . Combine t h i s with 2 parts I'M HC1 and 1 part 1% fas t green i n 95% ethanol. c) Feulgen 0 . 5 gms 100 mis Leucobasic fushsin ( S c h i f f ' s Reagent) H 20 ( d i s t i l l e d ) potassium metabisulphite 0 .5 gms basic fuchsin 10 mis 1. N HC1 35, Bring the water to a b o i l and then cool to 70°C. Add dye to the water and allow the solution to cool to room temper-ature. Let mixture stand f o r several hours i n a stoppered, brown b o t t l e . Add one gram decolourising charcoal and leave solution f o r 1/2 hour. Remove the charcoal by f i l t r a t i o n . Store the s t a i n at 4 C i n a brown bot t l e ( l i g h t sensitive s t a i n ) . Acid bisulphite wash 0.5 gms potassium metabisulphite 5.0 mis II N HC1 95.0 mis H 50 ( d i s t i l l e d ) Procedure: i ) dry c e l l s on albuminized s l i d e s i i ) f i x f o r 20 min. (ethanol: g l a c i a l acetic acid 3:1) i i i ) Hydrolyse f o r 11 min. at 60°C i n 31 N HC1 iv) rinse i n HgO ( d i s t i l l e d ) v) stain f o r 30-60 min. vi) rinse i n H 20 ( d i s t i l l e d ) for 10 min. v i i ) rinse i n acid bisulphite wash f o r 1 min. v i i i ) wash i n running water f o r 10 min. ix) counter s t a i n x) a i r dry xi) mount 36 4. Dryl's solution: Solution A: make stock to 0.1 M 5.88 gms sodium c i t r a t e 2.76 gms Na H"2 P0^ 2.84 gms Na 2 H PO^ Solution B: make 0.1 M stock . 2.22 gms CaCl 2 To make a 1 l i t r e solution: 20 mis sodium c i t r a t e 10 mis Na H 2 P0^ 10 mis Na 2 H P0^ 15 mis CaCl 2 Mix the f i r s t three solutions, add the d i s t i l l e d water to just below the 1 l i t r e mark, add the CaCl 2 and then:"add dis-t i l l e d water to the 1 l i t r e mark. Ph to 6.8. / 37 RESULTS I. Mutagenesis The chemical mutagen nitrosoguanidine (N-methyl-N'-nitro-N-Nitrosoguanidine) was used at a concentration of 75ug/ml fo r a period of 60 minutes. Seven separate mutagen-esis experiments were run and a t o t a l of 12 variants were recovered (Table I I ) . A relationship appears to exist be-tween the frequency of exautogamous death and the y i e l d of mutants (Table I ) . As the frequency of exautogamous death f a l l s below 45$, the y i e l d approaches zero; and as the f r e -quency of exautogamous death increases, there i s an increase i n the y i e l d of variants. The sudden increase i n the f r e -quency of exautogamous death i n experiments 6 and 7 (Table I) i was attributed to the use of a new preparation of nitroso-guanidine that was evidently more potent than the old prepar-ation. The frequency with which c e l l s underwent macronuclear regeneration at the time of selection was primarily caused by the cytot oxic effect of NTG and did not r e f l e c t differences of variant l i n e s . Presumably, the toxic effect of the mutagen caused abnormal development of the anlagen following autogamy, and, as a consequence, macronuclear regeneration occurred. i 38 The se l e c t i o n system used to recover these l i n e s was the same i n each experiment. This system was based on the fact that c e l l s undergoing macronuclear regeneration have a shortened c e l l cycle length at the f i r s t and second,cell cycles following nuclear reorganization. In wild type c e l l s , the f i r s t c e l l cycle following nuclear reorganization i s extended from f i v e hours (the normal vegetative c e l l cycle length)to 11 to 12 hours i n length. The second c e l l cycle i s a l s o extended to S-g-—9 hours (Berger, 1973b) . This increase in. the length of the c e l l cycle could be attributed to the time required f o r the new macronuclear anlagen to reach the DNA content of a mature macronucleus. C e l l s i n which the prezygotic macronuclear fragments have been released from the suppressive influence of anlagen accumulate DNA at a greater rate than do c e l l s undergoing normal nuclear reorgan-i z a t i o n (Berger, 1973a) . That i s , the amount of DNA synthes-ized by the t o t a l complement of regenerating fragments exceeds the amount of DNA synthesized by the growing macro-nuclear anlagen. Therefore, regenerating c e l l s should ar r i v e at the minimum DNA content required f o r c e l l d i v i s i o n before normally reorganizing c e l l s . Thus, the se l e c t i o n system involved the c o l l e c t i o n of early dividers, a r i s i n g i n p e t r i dish cultures, towards the end of the second c e l l cycle. Dividers could be found as early as 2 hours before the expected end of the second c e l l pycle. The recovered dividers were 39 placed i n i s o l a t i o n wells and allowed to complete the c e l l d i v i s i o n . One s i s t e r c e l l from each i s o l a t e was cloned, the other was dried on a microscope s l i d e , stained, and scored f o r the presence of regenerating macronuclear fragment For the f i r s t s i x experiments the selection was done following the f i r s t autogamy a f t e r mutagen treatment. In experiment 7, part of the selection was done following the f i r s t autogamy and part of i t was done following the second autogamy a f t e r mutagen treatment. The variants recovered a f t e r the f i r s t autogamy were those which come into phenotypi expression p r i o r to the f i r s t f i s s i o n of the exautogamont. This occurs because phenomic lag can be subst a n t i a l l y reduced by the starvation of exautogamonts.(Sonneborn, 1953; Berger, 1976). The mutagen-treated autogamous cultures were allowed to starve for a few days before - J r e - f eeding to allow any mutant geness to come into expression. In experiment 7, where the selection was done following the second autogamy, there was no change of genotype and thus no phenomic lag. It was hoped that selection at the second autogamy would reveal a wider range of variant phenotypes than would be obtained from se l e c t i o n a f t e r the f i r s t autogamy alone. TABLE I 40 MUTAGENESIS EXPERIMENTS: A COMPARISON OF THE FREQUENCY OF EXAUTOGAMOUS DEATH WITH THE YIELD EXPERIMENT NUMBER % VEGETA-TIVE DEATH % EXAUT-OGAMOUS DEATH (1) No. CELLS SCREENED YIELD (2) % M.R. AUTOGAMY Ov-1 20 40 1,500 1 3 i s t 2 20 50 1,500 3 8 1st 9 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 1st & 2n (1) The percent of exautogamous death following the mutagenesis i s an i n d i c a t i o n of the^frequency of l e t h a l mutations. (2) The y i e l d i s the number of variant c e l l l i n e s recovered. (3) T y p i c a l l y , selection was done following the f i r s t auto-gamy a f t e r the mutagenesis. In experiment 7 s e l e c t i o n was carried out a f t e r the f i r s t and second autogamy following mutagenesis. 41 TABLE II VARIANT LINES RECOVERED EXPERIMENT NUMBER LINE GENE PHENOTYPE 1 46-6c tam A - Missegregation of anlagen at nuclear reorganization - Abnormal d i s t r i b u t i o n of micro-nu c l e i at c e l l d i v i s i o n - Unequal d i v i s i o n , and mis-segregation of the macronucleus at c e l l d i v i s i o n - Trichocysts misshaped and do not discharge 2 1 0 -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 22-lc) - Abnormal nuclear events at conjugation - Source of macronuclear re-generating c e l l s - Source of amicronucleate c e l l s 7 16- 2c) 17- lb) - Slow growth 7 40-4b 1 tam G - Similar phenotype to tam A - Extra nuclei found following nuclear reorganization 7 40-4b 2 sp_ - Production of large, black c e l l inclusions i n the anterior part of the c e l l . 42 I I . Genetic Analysis of the Variants ' The genetic basis of the tarn A, tam. G and sp_ (spot) phenotypes were analysed by out-crossing to g e n e t i c a l l y marked wild type c e l l s . The F-^  phenotype was wild type with respect to a l l variant t r i a t s (tarn, sp. pw and nd). The loss of the pawn phenotype i n the 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. The Fg generation was obtained from F^ c e l l s by autogamy (Table I I I ) . In both tarn.A and tam G crosses the three t r a i t s , macronuclear missegregation, non-discharge of trichocysts and pawn, a l l segregated with a 1:1 r a t i o . There was complete concurrence of macronuclear missegregation and trichocyst non-discharge ( i n both tam A and tam G) suggesting that the genetic basis of both t r a i t s i s the same, single, recessive gene. S i m i l a r l y , sp and p_w segregated with a 1:1 r a t i o from t h e i r wild type a l l e l e s . Genetic analysis was not carried out i n l i n e s 10-la and 19-2b. Both of these l i n e s were ac c i d e n t a l l y k i l l e d when the c e l l s were fed toxic culture medium. Genetic analysis of l i n e 21-3c was attempted, but was not successful owing to the i n a b i l i t y of t h i s variant to complete conjugation successfully. The r e s u l t s of one attempt to outcross 21-3c to g e n e t i c a l l y marked pw A c e l l s are given i n table IV. From a t o t a l of 100 conjugating p a i r s not one conjugant successfully completed c r o s s - f e r t i l i z a t i o n . TABLE III A. Exautogamous segregation of tam A heterozygote, tam A/tam A+;pw A/pw A + B. Exautogamous segregation of tam G;sp double heterozygote, tam G/tam G+;pw A/pw A +;sp/sp' C. Exautogamous segregation of tam A;am double heterozygote, tam A/tam A+;pw A/pw A+;am/am' LINE F2 1 >HEN0TY] 3ES -REGOMB INANTS fo x 2 ( l : l ) 2 x 2 ( 3 : l ^ tam tam + A 1 tam A pw A tam A pw A+ tam A + pw A tam A + pw A+ .... 46-6 c 22 20 23 2$ 51.1. 0 .794 a 4 0 .0 b 42 48 B tam G sp. pw A tam G sp+ pw A+ tam G spj-pw A tam G sp pw A+ tam G + sp pw A tam G + sp+ pw A+ tam G + sp_ pw A+ tam G + sp+ pw A 40-4b 11 12 9 11 12 14 11 10 53.3 21 . 6 l c 0.O54d 43 47 6 tam A •"tarn A tam A tam A tam A f tam A + tam A + tam -A-- -am pwA am+ pw A+ am+ pw A am pw A+ am pw A am+ pw A+ am pw A+ am+ pw A 46-6 c d4-43 9 12 13 10 13 14 8 9 52.3 27.28e 0.375f 44 44 x c a l c u l a t e d u s i n g Y a t e s ' c o r r e c t i o n • 1. No s e p a r a t i o n between macronuclear missegregat ion and t r i c h o c y s t non -d ischarge t r a i t s was observed 2. E x p e c t a t i o n f o r a r e c e s s i v e a l l e l e at a s i n g l e l o c u s 3 . E x p e c t a t i o n f o r a double mutant a . P*=.90. b.Pe^less than . 0 5 , c . R ^ l e s s than . 0 5 , d .E*greater than .70 e . Pocless than . 0 5 , f . Ex g r e a t e r than .30 TABLE IV RESULTS OF MATING IN THE VARIANT 2 1 - 3c RESULTS NUMBER OF PAIRS Both conjugants dead 36 (36)* Cytogamy i n both conjugants 25 (25) Conjugants f a i l e d to mate 23 (23) 21-3c exconjugant dead 13 (13) pw A exconjugant dead 3 ( 3) C r o s s - f e r t i l i z a t i o n 0 ( - ) TOTAL 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 crossed to each other and to other tam and tam-like mutants to test f o r a l l e l i s m . In a l l cases the generation was wild-type f o r a l l t r a i t s [(table V). The mutation, pw A , was used as a marker i n the tam A and tam G parental macronuclei. Behaviour of the pw A marker indicated that 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 a l l cases. Tam A/tam A;jyti/am double mutant phenotype To determine whether the effects of the mutations were additive, tam A/tam A;am/am double homozygotes were recovered from the Fg progeny of a mating between tam A and am homozygotes. Approximately 25$ of the F^ progeny showed very slow growth, ( the mean generation time was 2 0 . 0 0 - 0 . 4 5 [ S . E . ] hours, compared with 7 . 3 7 -0 .20 [ S . E . ] hours f o r tam A/tam A and 6 . 2 5 - 0 . 1 6 [ S . E . ] hours f o r am/am). The slow growers displayed both macronuclear missegregation and trichocyst non-discharge t r a i t s and were assumed to be the double mutant, (table VI). The frequency of complete macronuclear missegregation i n the presumed double mutants did not d i f f e r from the frequency of missegregation observed i n c e l l s homozygous for either of the mutant genes alone. There was no effect of clonal age on the frequencyof macronuclear missegregation i n the double mutant. 46 TABLE V COMPLEMENTATION TEST BETWEEN TAM GENES GENE TAM A TAM G tam A + -tam 38 tam 33 - -tam 8 - -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 AGE (number of f i s s i o n s ) SAMPLE NUMBER •gam A/tam A; am/ am tam A/tam A am/ am + A 20 1 7/30(23)* 10/30(33) 2/30(7) 30 2 5/30(17) 6/30(20) 5/30(17) 50 3 11/30(37) . 14/30(47) 9/30(30) TOTAL 23/90(26) 30/90(33) 16/90(18] -() frequency as a percentage 48 I I I . Phenotypic Analysis A. Mutations a l t e r i n g the Pattern of Macronuclear D i v i s i o n  1) E f f e c t s on Vegetative C e l l s a) Generation time: The mean generation time of homozygous tam A c e l l s was 7.37±0.20[S.E.] hours as compared with 4.80±0.09[S.E.] hours f o r wild type. The mean generation time f o r tam G was s l i g h t l y shorter than f o r tam A, 6.29-0.27[S.E.] hours as compared with 5.00^0.11[S.E.] hours f o r the wild type control. The mean generation time f o r tam G i s very close to that calculated f o r am, 6 .25-0. l6[S.E.] hours. Probit analysis of the i n d i v i d u a l c e l l generation times of tam A reveals that the c e l l cycle lengths are not normally d i s t r i b u t e d (Figure 7 ) . Approximately h a l f of the c e l l s form a normally d i s t r i b u t e d subpopulation with a mean generation time of approximately 6.40 hours, and have the same variance as wild type controls. The upper part of the d i s t r i b u t i o n i s strongly skewed to the ri g h t , many c e l l s having very long generation times, and some, presumably amacronucleate c e l l s , do not divide at a l l . b) E f f e c t s on Shape and Size d i s t r i b u t i o n : A large percentage of tam A c e l l s have an abnormal FIGURE 7 . Probit analysis of tam A c e l l cycle lengths. wild* type tam A 50 a 3 N Q 3 N ' 51 f o o t b a l l shape compared to the oval s l i p p e r shape of wild type c e l l s (figure 8 ) . This difference i n shape i s seen to a lesser extent i n tam G c e l l s . There was substantial v a r i a t i o n i n the size of both di v i d i n g and morphostatic c e l l s i n cultures of both homozygous tam A and tam G c e l l s . Absorption microspectrpphpibicametric analysis of .Feulgen-stained and Napthol-Yellow counter-stained c e l l s showed that both cytoplasmic mass ( t o t a l protein) and macronuclear DNA content were approximately twice that f o r wild-type c e l l s . The mean postr-fission cumulative extinction f o r macronuclear DNA was 206.6i9.14 f o r tam A/tam A c e l l s ; 100.7-5.87 f o r wild-type c e l l s and 205.0-22.75 f o r the double mutant tam A/tam A; am/am. The cumulative extinction of t o t a l protein was 165.0^ 14 f o r tam A/tam A and 90- 16 f o r wild-type c e l l s . c) E f f e c t s on macronuclei i ) Phenotype The sequence of morphogenetic changes which the macronuclei of both tam A/tam A and tam G/tam G undergo during f i s s i o n was determined from a series of stained c e l l s f i x e d at various stages of f i s s i o n . The following analysis deals only with the most severely affected nuclei found at each stage of d i v i s i o n (figure 9 ) . Expression of both the tam A and tam G phenotypes i s variable and FIGURE 8. A comparison of c e l l morphology. 53 FIGURE 8 Figure 9 . Missegregation of the macronucleus at f i s s i o n i n c e l l s expressing the tam phenotype. Ma, macronucleus; mi, micronucleus; fv, 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 expressing the tam phenotype. B. Complete missegregation of the macronucleus at f i s s i o n i n c e l l s expressing the tam phenotype. FIGURE 9 56 almost h a l f the c e l l s i n populations of tam A/tam A and tam G/tam G cultures divide normally. By examining the most extreme phenotypes at each stage of c e l l d i v i s i o n i t was hoped that the sequence of events leading to e i t h e r p a r t i a l or complete missegregation of the macronucleus could be established. The macronuclei of homozygous tam A and tam G c e l l s migrate to the central part of the c e l l at about i n t e r f i s s i o n age 0.7. This corresponds to observations on wild-type controls. However, unlike wild-type c e l l s , the macro-nuclei of tam A/tam A and tam G/tam G c e l l s remain rounded and do not continue with the subsequent morpho-genetic events which occur i n wild type c e l l s (figure 10, plate 1). This lack of elongation and proper placement of the macronucleus of the tam mutants leads to varying degrees of unequal d i v i s i o n of the macronucleus ( p a r t i a l missegregation, plate 2) between s i s t e r c e l l s and, i n some cases, retention of the entire 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-segregation, plate 1, G). Where grossly unequal d i v i s i o n of the macronucleus occurs, the larger portion i s always retained by the anterior c e l l (proter), the posterior c e l l (opisthe) receiving the smaller portion (plate 3,D). Figure 1 0 . A comparison of macronuclear pr e d i v i s i o n morphogenesis between wild-type and homo-zygous tam c e l l s . ma, macronucleus; mi, micronucleus; fv, food vacuoles The decimal fracti o n s indicate the approximate i n t e r f i s s i o n age. 58 m a I 59 Complete missegregation of the macronucleus to the proter always occurs (plate 1,G; plate 2, A; plate 3, A). Macronuclear slippage has been observed i n other tam mutants, but was not observed i n eith e r tam A or tam G ( Ruiz, et a l . , 1976). In the most severe cases the macronuclei of homo-zygous tam A and tam G c e l l s do not divide at a l l , either during cytokinesis or immediately thereafter. Macronuclear elongation and c o n s t r i c t i o n i s e n t i r e l y absent. P a r t i a l missegregation of such a macronucleus r e s u l t s from the physical tearing of the undivided macronucleus by the advancing f i s s i o n furrow. In c e l l s expressing the tam phenotype to a lesser degree, s l i g h t elongation of the macronucleus i s evident, even while the macronucleus remains i n a central p o s i t i o n within the c e l l - while macronuclear c o n s t r i c t i o n may or may not occur. The slightly elongated macronucleus rounds up at the completion of cytokinesis. Misplaced macronuclei may elongate and c o n s t r i c t , but the c o n s t r i c t i o n may 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 separation of the two daughter c e l l s (plate 4, A). This can re s u l t i n the formation of small fragments of the macronucleus i n one or other of the s i s t e r c e l l s (plate 4, B and C). It i s not known whether these macronuclear fragments segregate at the 60 next c e l l d i v i s i o n and, i f they do, whether each becomes a f u l l y developed macronucleus. The pattern of missegregation of the macronuclei of homozygous tam A and homozygous tam G c e l l s i s not the same. Tam G shows a higher frequency of p a r t i a l missegregation and a lower frequency of complete mis-segregation when compared with tam A (Table VII). i'i) Penetrance and Expression a) Age E f f e c t s : (tam A only) Since the degree of macronuclear missegregation varied considerably between dif f e r e n t experiments, the age dependence' of phenotypic expression was examined. Age dependent expression had been demonstrated previously f o r a s i m i l a r mutant, am ( N o b i l i , 1959 and 1961). A series of c e l l l i n e s were started from exautogamonts. At approximately 10 f i s s i o n i n t e r v a l s samples of 100 dividers were recovered and stained, and the frequency of macronuclear missegregation was determined (Figures 11 and 12). The o v e r a l l pattern of macronuclear missegregation remained almost constant. However, examination of complete macronuclear missegregation alone revealed a change i n the frequency with clo 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 No. OF FISSIONS SAMPLE No. SAMPLE SIZE PARTIAL tam A tam G COMPLETE tam A tam G 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 Figure 11. Penetrance of the tam and am genes as a function of clonal age. A l l t r a i t s are included i n t h i s datum, i . e ; p a r t i a l and complete macronuclear missegregation, abnormal d i s t r i b u t i o n of micronuclei. V e r t i c a l bars indicate 95f° confidence i n t e r v a l s 63 Figure 12. The frequency of complete macronuclear mis-segregation i n tam' A/tam A and am/am c e l l s as a function of clonal age. •-• am/am (my data) A-A am/am (Nobili,1961) V e r t i c a l bars indicate 95% confidence i n t e r v a l s 6$ 66 the frequency of complete missegregation remained quite low (approximately 2 0 $ ) . At. 40 f i s s i o n s there was a dramatic increase i n the frequency of complete missegregation which dropped off only s l i g h t l y within the next 20 f i s s i o n s . The same experiment was carried out concurrently with homozygous am c e l l s f o r comparison. The degree of expression of tam A i s generally greater than that f o r am, e s p e c i a l l y i n young clones. Homozygous am c e l l s showed a greater age dependency than did homozygous tam A c e l l s . " ^ 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 either 34.5°C or 27.0°C f o r 24.00 hours. Dividing c e l l s from both temperature groups were collected, dried on s l i d e s , stained, and scored f o r both micronuclear and macronuclear missegregation (Table VIII). In tam G c e l l s , missegregation of the macronucleus did not occur at 34.5°C, while at the control temperature (27.0°C) 50$ macronuclear missegrggation occurred. The temperature effect was l e s s apparent i n homozygous tam A c e l l s . Missegregation of both types of nuclei occurred at 34»5°C and 27.0°C. However, the frequency of abnormal d i s t r i b u t i o n s of micronuclei and macronuclear missegregation was 10% higher at 27.0°C. 67 TABLE VIII THE EFFECT OF TEMPERATURE ON NUCLEAR MISSEGREGATION IN HOMOZYGOUS tam A AND tam G CELLS GENOTYPE FREQUENCY OF MACRONUCLEAR MISSEGREGATION FREQUENCY OF MICRONUCLEAR MISSEGREGATION 27.0 C 34.5 C 27 .0 C. 34.5 C tam A tam A 39/100(391 29/100(29) 26/100(26) 15/100(15) tam G tam G 17/34(50) 0/34(0) 5/34(15) 0/34(0) () frequency as a percentage 68 d) Ef f e c t s on Micronuclei The numbers of micronuclei observed i n tam A/tam A and tam G/tam G c e l l s varied considerably when compared to the number i n wild type c e l l s (Table IX). This i s c l e a r l y shown i n amicronucleate c e l l s where the micro-nuclei are not obscured i n the region of the macronucleus (Plate 5 ) . The d i s t r i b u t i o n of micronuclei at d i v i s i o n i n homozygous tam c e l l s was determined by analysis of the numbers of micronuclei i n related pairs of s i s t e r c e l l s (Figure 13). The number of micronuclei was compared between s i s t e r dividers and the parental micronuclear-complement was acsertained (Table X). The r e s u l t s are consistent with the hypothesis that abnormal numbers of micronuclei 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 analysis of the d i s t r i b u t i o n of micronuclei 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 either as early, as middle or as lat e dividers and the p o s i t i o n of the nuclei i n each c e l l was noted (Figure 15). Proper elongation of micronuclear d i v i s i o n spindles does not appear to occur i n tam A/tam A c e l l s and the micronuclei remain clustered i n the central region of the c e l l (Figure 16, Plate 3, A). Whereas i n wild type c e l l s , 69 TABLE IX COMPARISON OF THE NUMBER OF MICRONUCLEI BETWEEN HOMOZYGOUS tam A AND tam G CELLS AND WILD-,TYPE GENOTYPE NUMBER 2 OF ^2 MICRONUCLI >2 SI obscured + A 2 96/100(96) 1 - - 4/100(4) tam A^ tam A 30/75(40) 4/75(5) 29/75(39) 12/75(16) tam G^ -tam G 45/60(75) 0/60(0) ' 11/60(18) 4/60(7) 1) () frequency as a percentage 2) approximately 40 f i s s i o n s old 3) between 25-35 f i s s i o n s old I Figure 1 3 . Diagram of the experiment comparing the number of micronuclei i n related p a i r s of s i s t e r c e l l s . A. Isolated divider allowed to complete d i v i s i o n B. S i s t e r c e l l s separated and allowed to complete the next c e l l cycle (C and D) D. Pairs of r e l a t e d s i s t e r c e l l s dried i n microdrops on albuminized s l i d e s 7 1 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 SISTER DIVIDERS COMPLEMENT DISTRIBUTION ffam A tam G + OF PARENT BETWEEN tam A tam G + DIVIDER RELATED SISTERS (reconstructed) 2-2 2-2; ** 2-2 35/65(54) 78/120(70) 97/100 2-2 1-3, 2-2 4/65(6)* - -2-2 . 1-3; 1-3 4/65(6) 2/120(2) -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; ** 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 5-5 **• 5-5 1/65(2) 3/120(3) -6-6 6-6; 6-6** 1/65(2) - -1-1 1-1 •1-1 5/65J(8) 8/120(7) -1-1 1-1 > ** 2-0 2/65(3) - -J>() frequency as a percentage *Plate 6 Figure 14 . Theoretical diagram demonstrating how large numbers of micronuceli may aris e i n c e l l s expressing the tam phenotype. ma, macronucleus; mi, micronuclei; fv, food vacuoles Figure 1$. A. A comparison of early, middle and l a t e d i v i d e r s . B. Division of a d i v i d i n g c e l l into f i e l d s f o r the comparison of micronuclear l o c a l i z a t i o n , between tam/tam and wild-type d i v i d e r s . af, anterior f i e l d ; amf, anterior m i d - f i e l d cf, central f i e l d ; pmf, posterior m i d - f i e l d pf, posterior f i e l d FIGURE 15 Figure 16. I l l u s t r a t i o n of the difference i n micronuclear placement i n wild-type and tam A/tam A di v i d i n g c e l l s . ma, macronucleus; mi, micronucleus; fv, food vacuoles The decimal f r a c t i o n s indicate the approximate i n t e r f i s s i o n age j t a m t a m FIGURE 16 79 the spindles elongate and the micronuclei migrate towards the poles of the c e l l . This i s not true f o r a l l homozygous tam A and tam G c e l l s , but only f o r those that are expressing the tam phenotype. There was a s l i g h t temperature effect on the segregation of micronuclei, at 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 of missegregation was increased by 10% at 27.0°C. as compared to the observed frequency at j54.5°C. In homozygous tam G c e l l s the temperature effect was s l i g h t l y more pronounced. No micronuclear missegregation occurred at 34«5°C. as compared with a frequency of 14.5% at 27.0°C. e) Effect on Trichocysts The tri c h o c y s t s of both homozygous tam A and tam G c e l l s do not discharge when the c e l l s are k i l l e d with p i c r i c a c i d . Wild type c e l l s always discharge t h e i r t r i c h o c y s t s under these conditions. Observations on burst c e l l s , with negative phase contrast optics, reveals that abnormally shaped tr i c h o c y s t s are present i n the cytoplasms of tam A/tam A and tam G/tam G c e l l s (Figure 17). In homozygous tam A c e l l s , the morphology of the tricho c y s t s i s sim i l a r to that of the football-shaped variety described by Pollack (1974). Like the f o o t b a l l -Figure 17. I l l u s t r a t i o n showing the difference i n trichocyst morphology. A. Wild-type B. tam A/tam A C. tam G/tam G (The wild-type trichocysts are almost twice the length of the football-shaped and stubby trichocysts.) * i normal 3 8 , ooo football 32,000 stubby FIGURE 17 82 shaped trichocyst described by Pollack (1974), the abnormal tric h o c y s t s i n tam A/tam A c e l l s tend to dissolve when the c e l l i s burst. In preparations of burst wild-type c e l l s , the tr i c h o c y s t s do not dissolve. No surface or c o r t i c a l l y attached t r i c h o c y s t s are found i n tam A/tam A c e l l s which indicates a f a i l u r e of the trich o c y s t s to migrate within the c e l l ' s cytoplasm. In homozygous tam G c e l l s the morphology of the trichocysts i s sim i l a r to the stubby variety described by Pollack (1974)• Although no tric h o c y s t s are seen to discharge when c e l l s are k i l l e d with p i c r i c acid, a few trichocysts may discharge when c e l l s are burst under pressure. Stubby tric h o c y s t s do not dissolve, unlike the f o o t b a l l t richocysts of tam A/tam A c e l l s . 2) E f f e c t s During Nuclear Reorganization a) Autogamy: Both i n tam A/tam A and tam G/tam G c e l l s the nuclear events during autogamy are normal. The micronuclei undergo meiosis, a synkaryon i s formed from fusion of the two pro-n u c l e i , and the synkaryon completes two postzygotic d i v i s i o n s tbogive r i s e to the presumptive micronuclei and macronuclear anlagen. In wild type exautogamonts, segregation of the two macronuclear anlagen, without d i v i s i o n , occurs at the f i r s t f i s s i o n 83 following nuclear reorganization, restoring the vegetative nuclear complement of one macronucleus and two micronuclei per c e l l . In tam A/tam A and tam G/tam G'cells, however, both anlagen may segregate to one daughter c e l l at the f i r s t c e l l d i v i s i o n , leading to regeneration of the macronuclear fragments i n the other daughter c e l l (Figure 18b, Plate 7 , C,D, and E). In most cases where retention of both anlagen occurred i n one c e l l , i t was the proter. Although missegregation of anlagen occurred at the f i r s t f i s s i o n following nuclear reorganization, the occurrence was rare. More commonly, segregation of anlagen was normal at the f i r s t f i s s i o n , but missegregation of the macronucleus occurred with a high frequency at the second f i s s i o n . (Figure 18 a). Since the frequency of complete macronuclear missegregation during vegetative 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 considered that the frequency of missegregation of anlagen may also show an age dependency. That i s , the older the c e l l s are when they enter autogamy, the higher the frequency of mis-?-segregation of anlagen may be following nuclear reorganization. To test f o r t h i s p o s s i b i l i t y , cultures of homozygous tam A c e l l s of di f f e r e n t ages were starved and allowed to 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 comparing the frequency of macronuclear missegregation at, the f i r s t and second exautogamous d i v i s i o n . A. Complete missegregation of the macronucleus at the second exautogamous d i v i s i o n . B. Missegregation of one anlage at the f i r s t ' exautogamous d i v i s i o n . 86 into fresh medium and allowed to divide twice. After the f i r s t f i s s i o n , daughter c e l l s were separated. After the second f i s s i o n , two pairs of daughter c e l l s were dried on s l i d e s and stained with Azure A (Figure 18). The frequency of missegregation at 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 XI). Increased parental age does not lead to an increase i n the frequency of missegregation of macronuclear anlagen a f t e r autogamy. b) Conjugation: Homozygous tam A and tam G c e l l s form normal mating p a i r s . No unusual nuclear events were observed i n stained conjugating pairs of tam A/tam A c e l l s . Mis-segregation of macronuclear anlagen occurred at both the f i r s t and second f i s s i o n following conjugation. Abnormal nuclear events occurred occasionaMyjyin tam G/ tam G c e l l s giving r i s e to an excess number of micronuclei and/or macronuclear anlagen. In cases where excess numbers of nuclei were present, between 3 and 4 anlagen and 4 and 6 micronuclei were the most common complements. To determine what abnormal nuclear events occurring at conjugation were responsible f o r the excess numbers of nuclei i n exconjugants, a number of conjugating pairs were dried 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 No.OF NUMBER OF DIVIDERS SHOWING AGE CELL MISSEGREGATION OF ANLAGEN (f i s s i o n s ) LINES f i r s t f i s s i o n 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. It was thought that either the number of excess nuclei resulted from extra synkaryon d i v i s i o n s , or extra d i v i s i o n s of one or more of the synkaryon products, or that, the number of excess nuclei may r e s u l t from the persistence of some of the haploid meiotic products, which normally degenerate. Analysis of conjugating pairs showed that the proper number of haploid products (7) did not degenerate, but persisted through the stage of d i f f e r e n t i a t i o n of the synkaryon products. Among the conjugating pairs dried, and stained f o r analysis was one pa i r i n which an amacronucleate c e l l was coupled to a normal conjugant (Plate 3,C) i n a t y p i c a l mating configuration. Four small nuclei were observed i n the amacronucleate partner. Two of these nu c l e i were s l i g h t l y smaller and more f a i n t l y stained. These two appeared to be completing a d i v i s i o n (second synkaryon division? Or f i r s t hemikaryon d i v i s i o n ? ) . The remaining two nuclei were s l i g h t l y larger and more darkly staining (one placed a n t e r i o r l y , the other p o s t e r i o r l y placed, Plate 3,C). These two nuclei may be two of the haploid products that persisted through con- • jugation or they may be d i v i s i o n products of the synkaryon. Reference to amacronucleate conjugants has not been found i n the l i t e r a t u r e . 89 Occasionally, i n homozygous tam A or homozygous tam G exconjugants, development of certain products of the synkaryon was abnormal. In some cases one or both anlagen were undersized and f a i l e d to surpress DNA synthesis i n the macronuclear fragments (macronuclear regeneration i n evidence^ Plate 9,B). In other cases, normal sized anlagen were found i n the presence of early regenerating macronuclear fragments (Plate 9, Aand C). Exconjugant tam A or tam G c e l l s , undergoing macronuclear regeneration, underwent normal regeneration. The fragments aligned along the dorsal cortex, elongated during c e l l d i v i s i o n and segregated at c e l l d i v i s i o n , approximately one h a l f the number migrated to each s i s t e r c e l l (Plate 9,D). In some cases, missegregation of the regenerating fragments occurred. In such cases one s i s t e r c e l l received a l l or most of the fragments (usually the proter) while the other received no fragments or only one fragment (Plate 9,E). Regenerating fragments usually grow at approximately the same rate, so a l l the fragments within 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 regenerating fragments were of very, unequal size (Plate 9,F). 90 B. Variants with altered Micronuclear D i s t r i b u t i o n Lines 10-la. 19-2b and 21-3c  1) E f f e c t s on Vegetative c e l l s a) Generation time; The mean generation time f o r l i n e 10-la was 6.12^.08 [S.E.], f o r l i n e 19-2b was 5.35±.14[S.E.] hours, f o r l i n e 21-3c the mean generation time was 19.20-«26[S.E.] hours compared to 5 . 0 0 i.ll[S.E.] hours f o r wild type. The c e l l cycle length 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 wild type c e l l cycle length, whereas the delay i n the length of the c e l l cycle f o r the variant 21-3c was considerable. b) E f f e c t s on the Nuclei: ;l. v Division of the macronucleus was normal i n a l l three variants, with each daughter c e l l receiving approximately half of the p r e f i s s i o n macronucleus. P a r t i a l or complete macronuclear missegregation at c e l l d i v i s i o n was not observed i n any of these variants. Observations made on samples of stained i n t e r f i s s i o n c e l l s revealed a variable number of micronuclei, instead of the normal wild type complement of two. In l i n e s 10-la and 19-2b both abnormally high (5, 6 and 7) and abnormally low '§0 and.l) numbers of micronuclei were found (Table XII). In l i n e 2 l - 3 c , only 3.6% of the c e l l s contained the normal micronuclear 91 TABLE XII VARIATION IN THE NUMBER OF MICRONUCLEI IN MORPHOSTATIC CELLS OF LINES 10-la AND 19-2b LINE NUMBER OF MICRONUCLEI NUMBER 12 >2 10-la 49/75(65)* 8/75(11) 18/75(24) 19-2b 30/80(38) 11/80(13) 39/80(49) 51-s 94/96(98) 2/96( 2) -() frequency as a percentage 92 complement, 83.0$ had only a single micronucleus and 13.4$ had no micronuclei. It was considered that the excess micronuclei found i n l i n e s 10-la and 19-2b might arise through either extra di v i s i o n s of the micronuclei at the end of each c e l l cycle, or, that the extra numbers of micronuclei might aris e through missegregation of the micronuclei at c e l l d i v i s i o n . Analysis of stained, d i v i d i n g s i s t e r c e l l s allowed f o r the reconstruction of the micronuclear complement i n the parent d i v i d e r s . The r e s u l t s were consistent with the l a t t e r p o s s i b i l i t y ( T a b l e XIII). c)Penetrance and Expression: i ) Age E f f e c t s , ( l i n e s 10-la and 19-2b) T Since phenotypic expression i s often altered by physical or biochemical factors, the effect of age and temperature on expression 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 cloned. Samples of d i v i d i n g c e l l s were taken at 10 f i s s i o n i n t e r v a l s and the micronuclear complement was analysed (Figure 20). Line 10-la did not show age dependent expression. In l i n e 19-2b, however, the frequency of micronuclear missegregation increased i n c e l l s 50 f i s s i o n s old or older. There was an age dependent s h i f t from 93 TABLE XIII MICRONUCLEAR SEGREGATION AT CELL DIVISION IN PAIRS OF SISTER DIVIDERS MICRONUCLEAR MICRONUCLEAR NUMBER OF SISTER DIVIDERS COMPLEMENT SEGREGATION 10-la 19-2b -'51-S OF PARENT BETWEEN DIVIDER DIVIDERS 2-2 2-2 2-2 63/80(79) 81/112(72) 50/50(100) 2-2 1-3; 2-2 - 2/112(2) 2-2 1-3; 1-3 2/80(3) 3/112(3) 2-4 1-3 4-4 - 1/112(1) 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-•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) 1/112(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) 2/112(2) ' -4-4 4-4 4-4 2/80(3) - -4-4 4-4 ;2-6 2/80(3) - -5-5 5-5 5-5 — 2/112(2) () frequency as a percentage 94 extra micronuclei i n young c e l l s , to l e s s than two micronuclei i n older c e l l s , i n both l i n e s 10-la and 19-2b (Tables XIV and XV). i i ) Temperature E f f e c t s : The effect of temperature on the phenotype of l i n e s 10-la and 19-2b could not be examined as these l i n e s were l o s t before analysis of the phenotypes was completed. At 27.0°C and 34«5°C misdividers were found i n cultures of the variant 2l-3c. The frequency of misdividers-was low (between 2-5%). The misdividers formed two-cell monsters and occasionally, s h o r t - l i v e d , heteropolar doublets. 2) E f f e c t s During Nuclear Reorganization. a) Autogamy: Nuclear reorganization at autogamy was normal i n l i n e 10-la. No v a r i a t i o n i n nuclear complement was observed i n exautogamous c e l l s . A l l young clones had a high frequency (approximately 95%) of normal, vegetative c e l l s (two micronuclei). Variations i n the numbers of micronuclei and macronuclear anlagen was observed i n exconjugants and exautogamonts of l i n e 19-2b (Table XVI). Nuclear events at autogamy were not normal i n l i n e 21 - 3 c . i In a sample of 80 exautogamonts only 46.0% were vi a b l e . Of the remaining 54.0%, approximately 50.0% died within Figure 19. The Eff e c t of Age oh the Frequency of Micronuclear missegregation i n Lines 10-la and 19-2b. • - • 10-la m-* l9-2b 97 TABLE XIV NUMBER OF MICRONUCLEI AT DIVISION IN CELLS OF DIFFERENT AGES, LINE 10-la AGE SAMPLE SIZE No.AB-NORMAL' f 4-4 3-3 4-0 3-1 2-0 1-1 1-0 0-0 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 AGES, LINE 19-2b AGE SAMPLE No.AB-SIZE NORMAL $ 4 - 4 5 - 1 4 - 2 3 - 3 4 - 0 3 - 1 2 -0 1 - 1 1 -0 0 - 0 10 98 21 21 - - 1 2 1 5 4 - 2 -20 100 38 38 - - - 2 6 8 6 1 -30 60 22 37 1 1 1 10 - 3 - - - -40 100 36 36 - - - - 8 10 5 2 4 2 50 78 50 64 8 24 7 6 60 60 41 68 - - - - 1 - 2 26 5 4 99 TABLE XVI VARIATION IN THE NUMBER OF NUCLEI IN EXAUTOGAMOUS FIRST CELL CYCLE DIVIDERS, LINE 19-2b MICRONUCLEI MACRONUCLEAR TOTAL No. No. OF DIVIDING ANLAGEN OF SYNKARYON CELLS PRODUCTS -2-2 1 - 1 4(normal) 61/73 (84)* 4 - 4 1 - 1 6 2/73 ( 3) 2-6 1 - 1 6 1/73 ( 1) 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) *() frequency as a percentage 100 two days of completing autogamy without di v i d i n g , and approximately 3.0% completed one d i v i s i o n , a f t e r which both s i s t e r c e l l s died. From a sample of 100 stained exautogamonts, only 8% had completed nuclear reorganization with a normal complement of four n u c l e i , two micronuclei and two macronuclear anlagen. As many as 47.0% had no anlagen, and of those, almost 60.0% had no micronuclei (Plate 10, B and D). Of the remaining 52.0% that had either one or two anlagen, 44.0% had either a single micronucleus or none at a l l . Even when the normal nuclear arrangement of two micronuclei and two macro-nuclear anlagen were present, nuclear morphology was occasionally abnormal. In some cases the macronuclear anlagen were of very d i f f e r e n t sizes and the macronuclear fragments appeared to be shattered into t i n y s p l i n t e r s of chromatin (Plate 10,A). This observation on macro-nuclear disinte:gr.a#d;bni has also been observed i n amicronucleate Paramecium multimicronucleaturn ( D i l l e r , 1965). In a few cases, exautogamonts contained many micronuclei and small, darkly staining bodies, s l i g h t l y larger than micronuclei. It i s possible'that these bodies are grossly underdeveloped macronuclear anlagen (Plate 10 ,C). Exautogamonts containing only parental macronuclear fragments did not undergo macronuclear regeneration. The fragments remained small and f a i l e d to take on the 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 eventually died, either before 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 (Plate 11 A and B). Food vacuoles were usually absent (Plate 11,B), but occasionally one large food vacuole was present (Plate 11,A). b) Conjugation: (Line 21-3c only) Pair formation was i r r e g u l a r i n l i n e 21-3c. Couplets formed re a d i l y , but chains, closed and open t r i p l e t s , quadruplets and combinations of these were also formed (Figure 20). Nuclear reorganization occurred i n most of the c e l l s present i n a clump or chain, 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 conjugation. In some cases, members of a clump or chain retained the vegetative nuclear arrangement,- while other members of the same clump or chain were engaged i n nuclear r e-organization. In any culture of c e l l s belonging to the l i n e 21-3c many amicronucleate exautogamonts were present. When cultures containing amicronucleates were mixed with c e l l s of 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 of the amicronucleates was already fragmented, no futher nuclear changes was observed i n the amicronucleate conjugants. The normal partner of an j 102 amicronucleate mating p a i r usually underwent the t y p i c a l nuclear changes of conjugation. In most cases, both exconjugants from an amicronuclate sexual arrangement were nonviable. Observations on stained conjugating pairs of 21-3c x wild type c e l l s revealed two abnormalities i n the nuclear behaviour of the 21-3c conjugant. F i r s t l y , meiosis i n the partners of a mating pair was asynchronous (Plate 12, A and B). The partner belonging to l i n e 21-3c always appeared to be a pregamic d i v i s i o n behind the wild type partner. 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 pronuclei did not take place. Secondly, the positioning of the haploid meiotic products was often abnormal. In most cases, i n the wild type conjugant, the meiotic products are c e n t r a l l y located i n the conjugant, near the region, of the paroral cone, while the fragments of the prezygotic macronucleus are peripheral, surrounding the haploid pronuclei. In conjugants belonging to the l i n e 21-3c, the fragments of the prezygotic macronucleus are scattered throughout the conjugant c e l l , the pronuclei seldom l i e near the region of the paroral cone, but l i k e the fragments, are also scattered about the c e l l . I Figure 20. Anormal Mating Configurations from a WildnType Cross to Line 21-3c C e l l s . I 104 FIGURE 20 105 DISCUSSION Genetic analysis of the two tam mutants, tam A and tam G ? reveals that the abnormal nuclear events both at c e l l d i v i s i o n and at nuclear reorganization, 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 single, recessive gene i n each mutant. Separation of abnormal nuclear events and abnormal trichocyst 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 effect of the tam gene ac t u a l l y being caused by two or more c l o s e l y linked mutations seems un l i k e l y since several s i m i l a r mutants have f a i l e d to show separation of the d i f f e r e n t aspects of the tam and tarn-like phenotypes (Sonneborn, 1975; Ruiz, et a l . , 1976). In only one of 18 tam-like mutants i s there independent determination of abnormal nuclear t r a i t s and abnormal trichocyst morphogenesis. These t r a i t s are separately 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, the re i s a p a r t i a l b lockage of t r i c h o c y s t d ischarge (Aufderhe ide , p e r s o n a l communicat ion) . Despi te t h i s one p a r t i a l e x c e p t i o n , i t i s c l e a r t h a t 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, t r i c h o c y s t 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 the occurrence of l a r g e , b l a c k , v e s i c u l a r , c r y s t a l l i n e , cy top lasmic i n c l u s i o n s ( s p o t s ) . Genet ic a n a l y s i s of Fg c e l l s from a c r o s s of t h i s s tock t o w i l d - t y p e , showed independent assortment of the tam phenotype' and the 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 mutat ions of two separate genes, tam G and s p . L inkage of the tamG and sp_ genes appeared u n l i k e l y s i n c e recombinat ion between them was 4 3 . 0 - 1 0 % (Ri=' .50) . Genet ic a n a l y s i s of v a r i a n t 21-3c was not p o s s i b l e because c e l l s be long ing t o t h i s l i n e d i d not complete c o n j u g a t i o n . 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 before complet ion of the g e n e t i c a n a l y s i s . A comparat ive 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 shows tha t the 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 are 107 longer than that of wild type c e l l s . The degree to which the c e l l cycle i s lengthened seems to correspond to the degree of divergence of the variant phenotype from the wild type phenotype. For example, the c e l l cycle lengths i n the more severely abnormal phenotypes (tam A and tam G) are extended by approximately three hours and two hours respectively, while the variant sp, which d i f f e r s only s l i g h t l y i n phenotype from wild type c e l l s , v . extends the c e l l cycle length only 20 minutes. Extensions i n c e l l cycle lengths seem to be common to most mutants expressing non-wild type phenotypes i n Paramecium (see Sonneborn, 1975). C e l l - s i z e , macronuclear-size and the length-to-width r a t i o seem to have been altered i n c e l l s homozygous f o r either tam A or tam G. I n t e r f i s s i o n c e l l s and d i v i d i n g c e l l s were as much as one and one-half times the length of wild type c e l l s at the same stage. This change i n c e l l size i s not 100$ penetrant, but appears only i n c e l l s expressing the tam phenotype. Moreover, tam A/ tam A c e l l s grown at 17.0°C and/or starved f o r several days appear to readjust to a normal size (approximately 120um x 50um). Absorption microspectrophotometric measurements on Napthol Yellow stained G-, c e l l s , reveals I 108 that protein content i s almost twice that of wild type controls. There appears to be a positi v e c o r r e l a t i o n between c e l l size and t o t a l protein. This i s i n agreement with observations made by Jauker (1975) i n Tetrahymena. The problem of explaining t h i s r e l a t i v e l y stable doubling i n cytoplasmic mass was not resolved during t h i s study. The observed extention i n the length of the c e l l cycle i n tam A/tam A and tam G/tam G c e l l s could influence c e l l s i z e . Jauker (1975a and b) found that the gross protein content per c e l l appeared to be a function of the generation time, within l i m i t s . He found that the l i n e a r i t y of t o t a l protein as a function of generation time was upset a f t e r very long c e l l cycle lengths. This observation of Jauker's could explain why the double mutant .am/am;tam A/tam A, which has a c e l l cycle length of four times that of wild-type c e l l s , was not correspondingly enlarged. The double mutant am/am;tam A/tam A. l i k e tam A/tam A. i s only approximately twice the size of wild-type control c e l l s . Since c e l l size and macronuclear DNA content remain within cer t a i n l i m i t s , some type of regulation process must e x i s t . Kimball (1967) demonstrated that i n e q u a l i t i e s i n cytoplasmic mass and DNA content, introduced at c e l l d i v i s i o n , are reduced over the following c e l l generations, ' both i n wild-type 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 stable change i n c e l l and macronuclear size seen i n tam A/tam A c e l l s does not appear to be subject to regulation i n the same way. It i s therefore suggested that factors other than an extension i n generation time are i n f l u e n t i a l i n perpetuating these size differences. One possible explanation i s the occurrence of an interruption i n the normal state of equilibrium that exists between c e l l p r o l i f e r a t i o n and c e l l growth rate. This possible s h i f t i n the equilibrium would begin when certain c h a r a c t e r i s t i c s influenced by the tam gene come into expression. For example, r a i s i n g the temperature from 17.0°C to 27.0°C might i n i t i a t e an increase i n c e l l growth rate while c e l l d i v i s i o n rate would not undergo a si m i l a r increase immediately, i n compensation. By the time the rate of c e l l d i v i s i o n catches up with the rate of c e l l growth, restoring an equilibrium between the two variables, the c e l l s have attained a size almost twice that of t h e i r wild type counterparts. These r e l a t i v e l y stable changes i n c e l l size must occur during c e l l d i v i s i o n , since i t has been suggested that the number of c o r t i c a l units per c e l l changes only at t h i s time (D. Jones, 1977). I n t e r f i s s i o n c e l l s may change i n size , but i t might be expected that i n t e r f i s s i o n c e l l size changes are li m i t e d by the l i m i t e d f l e x i b i l i t y i n the size of the c o r t i c a l u n i t s . The stable 110 " change i n macronuclear DNA content i s independent of the v a r i a t i o n introduced at c e l l d i v i s i o n through the action of the tam gene. It' i s suggested that t h i s stable doubling of the macronuclear DNA content i s a dir e c t consequence of the change i n cytoplasmic mass. That the amount of macronuclear DNA i s proportional to the mass of the c e l l has been suggested by Berger (1977). This rel a t i o n s h i p appears to be coupled i n a one-way i n t e r a c t i o n (Berger and Schmidt; 1977; Morton and Berger, 1977; Berger,1977). That i s , changes i n protein content do not occur i f DNA content changes, but i f t o t a l protein content changes, the macronuclear DNA content adjusts accordingly. The one-way coupling of cytoplasmic mass and DNA content reveals that the nucleo-cytoplasmic r a t i o between them i s not ar-ei?it-ie.al precondition f o r f i s s i o n , as has been suggested f o r Tetrahymena, (Worthington, et a l . , 1976). This i s an important fact which can explain how r e l a t i v e l y stable changes i n c e l l size can occur through changes i n growth rate without concommitant r e l a t i v e changes i n the d i v i s i o n rate, which would necessarily correct changes i n nucleo-cytoplasmic r a t i o s . In addition to the r e l a t i v e l y stable change i n c e l l size and mean macronuclear DNA content i n tam A/tam A I l l c e l l s , further changes i n DNA content are introduced at f i s s i o n through the action of the tam gene. These changes are not r e l a t i v e l y stable, but appear to be subject to regulation (Berger and Schmidt, 1977). It has been found that the variance of induced nuclear DNA content from the population mean, i s reduced by one half i n a single c e l l cycle, and that the variance continues to be reduced by one h a l f i n each successive c e l l cycle (Berger and Schmidt, 1977). Although the mean c e l l cycle length i n l i n e 2l-3c i s almost four times the length of that of wild type c e l l s , the average c e l l size i s equal to or smaller than that of wild-type c e l l s . This can be attributed to the fact that the majority (83.0%) of c e l l s i n any culture of 21-3c are amicronucleate exautogamonts. Feeding appears to be a major problem i n such c e l l s . This suggestion i s based on observations on stained c e l l s which reveal an absence of food-vacuoles i n the majority of amicronuclate c e l l s . These amicronuclate c e l l s may pe r s i s t f o r a few days, but few f a i l to complete an entire c e l l cycle. Replacement of the or a l apparatus and ventral c i l i a t i o n apparently occurs at conjugation, i n Paramecium (Roque, 1956). It has been suggested that 1 1 2 replacement of the or a l apparatus at conjugation, i n amicronucleate c e l l s , i s incomplete ( D i l l e r , 1 9 6 5 ) . Skoblo ( 1 9 6 9 ) found that amicronucleate Paramecium  caudatum exconjugants could not feed, and Ossipov and Tavrovskaya ( 1 9 6 9 ) found that amicronucleate exconjugants showed no signs of forming a new g u l l e t i n P. caudatum. It i s possible, therefore, that regeneration of the or 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 that 1 ) the macronuclear envelope remains intact through-out karyokinesis, 2 ) a mitotic apparatus i s not present i n the d i v i d i n g macronucleus and 3) there i s no condensation of chromosomes. Macronuclear d i v i s i o n i s accomplished through simple c o n s t r i c t i o n of the macro-nucleus into approximately two equal parts. Although a t y p i c a l mitotic spindle i s not present i n the d i v i d i n g macronucleus, intranuclear microtubules, and bundles of microtubules associated with the nuclear membrane have been observed i n many c i l i a t e s (Tucker, 1 9 6 7 ; Suganuma, 1 9 6 9 ; Roth and Minick, 1 9 6 1 ; Tamura, ejp a l . , 1 9 6 9 ; Jurand and Selman, 1 9 7 0 ; Inaba and Kudo, 1 9 7 2 ) . Despite the many c y t o l o g i c a l observations on a m i t o t i c a l l y d i v i d i n g 113 macronuclei, the mechanisms governing the morphogenetic events are not known. These morphogenetic events are severely altered i n c e l l s expressing the tam phenotype. The macronuclei of tam A/tam A and tam G/tam G c e l l s undergo the very early p r e d i v i s i o n events and reach a central p o s i t i o n i n the pre d i v i s i o n c e l l . However, the macronuclei i n these c e l l s forgo any further morpho-genesis, they f a i l to reach the normal subcortical l o c a t i o n on the dorsal side of the c e l l , they f a i l to elongate properly and macronuclear c o n s t r i c t i o n does not take place. This i n a b i l i t y of the macronucleus to complete pr e d i v i s i o n morphogenesis has been observed i n other tam mutants, notably tam 6, tam 8 (Beisson and Rossignol, 1975) and tam 38 (Ruiz, et a l . , 1976). There are several possible explanations that could account f o r the abnormal behavior of the macronucleus i n homozygous tam c e l l s . Failure of the macronucleus to complete pr e d i v i s i o n morphogenesis ' may be attributed to: 1) improper cytoplasmic l o c a l i z a t i o n of the macronucleus; 2) improper polymerization of i n t r a -nuclear and/or extranuclear microtubules and 3) defective membrane attachment s i t e s . A series of gra f t i n g experiments on Stentor lends support to the f i r s t , possible explanation. These \ 114 experiments reveal that cert a i n aspects of macronuclear behavior are influenced by c o r t i c a l signals (de Terra, 1971 and 1973). The c o r t i c a l signals have a very short range of influence and, therefore, can only act on su b c o r t i c a l l y located macronuclei (de Terra, 1971 and 1973). I f such a system of c o r t i c a l influence exists i n Paramecium, and i f , as i n Stentor, these signals have a very short range, then, i t may be suggested, that the misplaced macronucleus f a i l s to receive the signals that morphogenesis should proceed. Several references lend support to the second p o s s i b i l i t y , that 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 to continue with pred i v i s i o n morphogenesis, such as the f a i l u r e to elongate. Tamura, e_t aJL., (1969) found that the macronuclei of c e l l s treated with colchicine, a drug known to in t e r f e r e with microtubule polymerization f a i l e d to elongate properly: improper placement of the macronucleus was observed, and, i n c e l l s that completed nuclear d i v i s i o n , the d i v i s i o n of the macronucleus was unequal. Williams and Williams (1976) reported that c e l l s treated with colchicine just p r i o r to macronuclear elongation, completed d i v i s i o n , but they found that 115 macronuclear morphogenesis was abnormal. The macronuclei i n treated c e l l s , f a i l e d to elogate, nuclear c o n s t r i c t i o n did not occur, the macronuclei remained i n a central location and nuclear morphology was abnormal. Electron microscopic examination of these colchicine treated c e l l s revealed a complete absence of the intranuclear and membrane associated microtubules. Ruiz, .et a l . , (1976) reported the induction of phenocopies of the mutant tam 38 by tr e a t i n g wild-type c e l l s with either colchicine or v i n b l a s t i n . Further evidence f o r the possible involvement of microtubules i n macronuclear elongation and c o n s t r i c t i o n comes from a study by Walker and Goode (1976) on the role of microtubules i n macronuclear d i v i s i o n i n two hypotrichs, Gastrostyla and Stylonychia. The authors found that the macronuclei of c e l l s treated with 60.0% deutrium oxide, an agent known to hyperpolymerize microtubules, were hyperextended, exhibited abnormal morphology, and often f a i l e d to complete karyokinesis. However, agents other than those d i r e c t l y a f f e c t i n g microtubule assembly, can also induce s i m i l a r , abnormal events i n ^ PK©<iivision macronuclear morphogenesis. C e l l s treated p r i o r to, or at i n t e r f i s s i o n age 0.7, with 116 actinomycin D (a drug known to i n h i b i t RNA synthesis) f a i l to divide ( G i l l and Hanson, 1976) . C e l l s treated a f t e r i n t e r f i s s i o n age 0 .7 completed cytokinesis, but i n many cases cytokinesis was not accompanied by karyokinesis - the i n d i v i d u a l p r e f i s s i o n macronucleus was retained i n the proter. G i l l and Hanson (1976) also found that, where cytokinesis was accompanied by karyokinesis, i n actinomycin D treated c e l l s , macro-nuclear d i v i s i o n was unequal and c e l l s receiving the smaller amount of DNA were in v i a b l e . Examination of the actinomycin D treated c e l l s revealed large aggregates of p r o t e i n - l i k e material which G i l l and Hanson (1976) suggested may be related to microtubule formation. The authors did not off e r an explanation as to how actinomycin D was a f f e c t i n g microtubule polymerization. The p o s s i b i l i t y remains that any toxic drug w i l l i n t e r f e r e with p r e f i s s i o n macronuclear morphogenesis . There i s a suggestion, however, that actinomycin 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 assembly factor (Bierber, 1972) . Although wild-type c e l l s , treated with anti-micro-tubule drugs mimick the phenotype produced by tam mutations, i t does not mean that the tam l e s i o n d i r e c t l y 117 a f f e c t s the microtubule population. The a b i l i t y of microtubules to 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 to r a p i d l y regenerate shed c i l i a . In addition, i t i s d i f f i c u l t to imagine what possible effect microtubules would have i n influencing trichocyst morphogenesis. The t h i r d possible explanation i s that the tam l e s i o n a f f e c t s the plasma membrane and/or the macro-nuclear envelope. One p o s s i b i l i t y i s that the tam mutation leads to a disruption of intramembraneous p a r t i c l e arrays, which would obscure attachment-sites normally available to 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 microtubules (or microfilaments) to make an attachment would be compatible with the apparent incompetence of the microtubules, and the a b i l i t y of anti-microtubule drugs to mimick the tam phenotype. The p o s s i b i l t y of a disordered membrane ex i s t i n g i n homozygous tam c e l l s can more r e a d i l y be applied as the basiespsrobil-em'*under-l y i n g the p l e i o t r o p i c effect of the tam gene. Intra-membraneous microtubule bridges are known to exist (see 118 Al l e n , 1975) . In addition there i s evidence to support the idea that microtubules are involved i n membrane-bound, 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 r e l o c a l i z a t i o n of a nucleus. Holmes and Choppin, 1968; Messier and Auclair, 1973; Robison and Charlton, 1 9 7 3 ; Wagner and Rosenberg, 1973; A l l e n , 1974) , and evidence that supports the idea of extranuclear microtubule association with the macronuclear envelope i n many c i l i a t e s (Roth and Shigenaka, 1964; Schulster, 1965; Jenkins, 1968; Inaba and Sotokawa, 1968) . It may be suggested, therefore, that i n the wild-type c e l l , •microtubule linkages form between the plasma membrane and the macronuclear envelope, guiding the macronucleus to a subcortical location and f i r m l y anchoring the p r e f i s s i o n macronucleus to the cortex, i n preparation f o r macronuclear elongation. Thus one could speculate that these microtubule linkages f a i l to form i n homozygous tam c e l l s due to the i n a b i l i t y of the micro-tubules to recognize the alte r e d membrane attachment-s i t e s . Consequently, r e l o c a t i o n of the p r e f i s s i o n macronucleus and further morphogenesis does not take place. Experimental support f o r t h i s idea exists i n the l i t e r a t u r e pertaining to trichocyst morphogenesis and trichocyst attachment, and w i l l be discussed l a t e r . Since the degree of macronuclear missegregation varied between di f f e r e n t experiments, the possible influences of age and temperature on penetrance and expression of the tam gene were examined. N o b i l i (1961) had found that a s i m i l a r mutant, am/am, showed age-dependent expression. Only homozygous tam A c e l l s were examined f o r age-dependent expression. When a l l the aspects of the tam gene were considered there was no age-dependent expression i n tam A/tam A i n d i v i d u a l s . However, complete macronuclear missegregation, when examined alone, showed a d e f i n i t e increase i n frequency with increasing clonal age. At the same time, p a r t i a l macronuclear missegregation showed a decrease i n frequency with increasing age of the clone. Thus, i t appeared that the more severe form of macronuclear mis-segregation increased i n older clones. Expression of mutations of both tam A and tam G showed a s l i g h t temperature dependency. Macronuclear missegregation increased i n frequency at 27.0°C when compared with the frequency at 34.5°C. I f microtubule polymerization i s the basic problem behind abnormal macronuclear morphogenesis, then one could speculate that the rate of microtubule polymerization might be 120 increased at the higher temperature. It could also be suggested that changes i n membrane-bound p a r t i c l e arrays occur at the higher temperature, thus unmasking possible attachment-sites. A l t e r n a t i v e l y , the tam genes may be cold sensitive,genes with impaired function at lower temperatures. 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 occurs both i n c e l l s expressing the tam phenotype and i n c e l l s belonging to the l i n e s 10-la-and 1 9 - 2 b . From c y t o l o g i c a l observations on stained c e l l s , i t appears > that the micronuclei f a i l to migrate to t h e i r normal loc a t i o n at the poles of the d i v i d i n g c e l l . Instead, the micronuclei remain clustered around the plane of cleavage and are randomly d i s t r i b u t e d by the advancing f i s s i o n furrow. This frequently leads to errors i n the 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 . Improper cytoplasmic l o c a l i z a t i o n appears to be the basis of both micronuclear and macronuclear abnormal behaviour patterns i n homozygous tam c e l l s . The fact that other tam mutants also exhibit i r r e g u l a r d i s t r i b u t i o n of micronuclei would suggest the p o s s i b i l i t y of a coupling between the two types of nuclear behaviour t r a i t s (see Ruiz, et a l . , 1976) . However, i f some sort of 121 coupling does exi s t , i t cannot be very t i g h t , since i r r e g u l a r d i s t r i b u t i o n s of micronuclei can occur i n the absence of macronuclear missegregation ( l i n e s 10-la and 19-2b). Two hypotheses are suggested to explain the observed misplacement of micronuclei 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 micronuclei may arise from the f a i l u r e of the mitotic spindle to elongate (a p o s s i b i l i t y also suggested by Ruiz, e t _ a l . , 1976). Secondly, micronuclear mis-placement may be due to the f a i l u r e of membrane bridges or microtubule linkages to form between the macronucleus and the micronuclei. In £. t e t r a u r e l i a , the intranuclear mitotic spindle forms a separation spindle, pushing the daughter micronuclei away from the l a t i t u d e of cleavage, ;' towrds the poles of the di v i d i n g c e l l (see Tucker, 1967). Thus, inadequate elongation of the separation spindle could r e s u l t i n the lack of movement of the daughter micronuclei and the subsequent abnormal d i s t r i b u t i o n of the micronuclei at f i s s i o n . There i s l i t t l e evidence i n the l i t e r a t u r e to support the 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 kind was made by Tucker (1967) i n the c i l i a t e Nassula. The form of linkage i n t h i s species consists of membrane bridges connecting the di v i d i n g macronucleus with the m i t o t i c a l l y d i v i d i n g micronuclei. Tucker suggests that the bridges may be important i n assuring even d i s t r i b u t i o n of micronuclei to s i s t e r c e l l s . He. went on to speculate that i f these membrane bridges were absent and the daughter micronuclei were free to move through the cytoplasm, they might be unequally di s t r i b u t e d between s i s t e r c e l l s when cytokinesis was completed. In Paramecium multimicronucleatum a d i f f e r e n t type of macronuclear-micronuclear linkage has been observed (Inaba and Kudo, 1972). In t h i s linkage system microtubules are found to connect the early d i v i d i n g macronucleus with the m i t o t i c a l l y d i v i d i n g micronuclei. However, the microtubules are not seen a f t e r micronuclear metaphase. The suggested role f o r t h i s microtubule l i n k between the macronucleus and the micronucleus, i s that the microtubules separate the micronuclei from the macronucleus during the early stage of macronuclear d i v i s i o n . In Paramecium bursaria, extranucleaas microtubules have been seen to connect the micronucleus to the c e l l surface during metaphase and through anaphase of micronuclear d i v i s i o n (Lewis, et a l . , 1976). The authors did not 123 discuss the possible significance 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 that these microtubule linkages may account f o r equal d i s t r i b u t i o n of micronuclei to daughter nuclei at f i s s i o n . Theoretically, i r r e g u l a r micronuclear d i s t r i b u t i o n could eventually give r i s e to c e l l s with multi-micronuclei (10, 16 and many more), and yet no more than six micronuclei have been observed i n any one i n t e r f i s s i o n c e l l . This suggests either that c e l l s with a very high micronuclear complement have reduced v i a b i l i t y , or that some sort 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 regulation of micronuclear number exists i n Gastrostyla s t e i n i i . Walker (1976) observed that not a l l four micronuclei took part i n mitosis at any given c e l l d i v i s i o n or at excystment. He found there was an i n t r a c e l l u l a r discrimination between the micronuclei and that one or more may forgo d i v i s i o n . This asynchronous behaviour of micronuclei i n Gastrostyla may be away of regulating micronuclear numbers. Since numbers of micronuclei have been observed i n stained cells-, that are not multiples of two and, that there exists an age dependent s h i f t toward lower numbers of micronuclei, the p o s s i b i l i t y remains that P_. t e t r a u r e l i a can control 124 the number of micronuclei embarking on mitosis i n any one c e l l cycle. The trichocysts of homozygous tam c e l l s are unable to discharge, they are not attached to the c e l l ' s surface and they have an abnormal morphology ( f o o t b a l l -shaped i n tam A/tam A c e l l s and stubby i n tam G/tam G). The normal trichocyst cycle begins with morphogenesis inside small, cytoplasmic v e s i c l e s . The f u l l y formed organelle then migrates to the c e l l ' s cortex where i t attaches at s p e c i f i c s i t e s on the plasma membrane. Exo.cytosis i s the f i n a l event i n the trichocyst cycle. Both genes, tam A and tam G, a f f e c t trichocyst development very early i n the cycle. Detailed examination of f o o t b a l l -shaped tric h o c y s t s reveal that a number are t i p l e s s , but they may have unattached, abortive t i p s l y i n g beside the organelle, within the v e s i c l e (Pollack, 1974) . A very few have misshapen tips, that are attached (Pollack, 1974) . Stubby trichocysts have a variable morphology, a few may be attached to the c e l l ' s cortex; i n some, a small segment of the t i p and some sheath material are located along side the organelle (Pollack, 1974) . In twelve other tam and tarn-like mutants, abnormal trichocyst morphogenesis i s apparent i n addition to abnormal nuclear behaviour. 125 In f i v e other mutants, nd 6 , nd 9, nd 3. nd 7 and pt 2 , (Ruiz, e t _ a l . , 1976) i n which trichocysts f a i l to discharge, there i s no evidence of abnormal nuclear behaviour. In these f i v e l a t t e r mutants, tric h o c y s t s are capable of attachment to the plasma membrane, whereas i n a l l the other s i m i l a r mutants, where both trichocyst and nuclear t r a i t s are i n evidence together, no trichocyst attachment i s observed. On the basis of the apparent c o r r e l a t i o n between trichocyst attachment and nuclear behaviour, Ruiz, .et _al. (1976) suggest that the nuclear abnormalities common to t h e i r tam mutants are d i r e c t l y due to the absence of attached t r i c h o c y s t s . They further suggest, that a l l mutants with unattached tri c h o c y s t s should have abnormal nuclear d i v i s i o n s . This theory, put forward by Ruiz, et a l . (1976) would f i t with the idea that trichocyst and nuclear morphogenesis are linked to the same biochemical pathway, or comprise two major branches of one pathway. I f t h i s were the case the trichocyst branch would be d i s t a l to the nuclear branch. Thus, one could anticipate independent trichocyst mutations, but nuclear abnormalities would always be associated with trichocyst abnormalities. The most d i f f i c u l t part of discussing the tam gene i s f i n d i n g a common denominator between the trichocyst 126 t r a i t and the nuclear t r a i t . It i s d i f f i c u l t to imagine that a v i t a l i n c l u s i o n such as the nucleus, ^should be so t i g h t l y coupled to a system of organelles, the t r i c h o c y s t s , lacking any obvious function. The rela t i o n s h i p might be very subtle or i t might be something as obvious as microtubules, microfilaments or membrane attachment s i t e s . This study provides no explanation of the.pleiotropic nature of the tam gene. The most plausible suggestion i s that membrane-bound pa r t i c l e . a r r a y s may be abnormal i n c e l l s expressing the tam phenotype. This could then res u l t i n the lack of formation of re-cognizable attachment s i t e s f o r trichocysts as well as microtubules. I f t h i s were the case, one could anticipate that organelle movement would be impaired, which i s the basic problem observed i n homozygous tam A and tam H c e l l s . In support of t h i s idead i s the fact that s p e c i f i c plasma membrane s i t e s have been i d e n t i f i e d f o r trichocyst and; i n Tetrahymena, mucocyst attachment. The trichocyst attachment s i t e s are severly altered i n t l , tam 8 and nd_9 (Beisson, et a l . , 1976). Tr'lchocyst membrane attachment s i t e s appear as an ordered array of p a r t i c l e s arranged i n an inner rosette and an outer r i n g (Janish, 1972; S a t i r , _ e t a l . , 1972; Plattner, et a l . , 1973; Beisson, e t _ a l . , 1976). Beisson et al.- (1976) found that the outer r i n g of p a r t i c l e s i s always present, even i n mutants 127 containing no tri c h o c y s t s ( t l ) . In mutants containing unattached trichocysts or no tric 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 collapsed, forming a parenthesis. In c e l l s with attached t r i c h o c y s t s , the outer rings are c i r c u l a r (nd9 and wil d type). The authors suggest that the collapsed rings correspond to 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 to f i l l e d s i t e s . In addition, Beisson, et a l . (1976) found that the inner rosettes are only present when trichocysts are attached, and that they appear to be an e s s e n t i a l feature of trichocyst discharge ( i n nd9 at 27.0°C, there i s trichocyst attachment but no discharge; the rosettes are abnormal. At the permissive temperature^ the rosettes recover and the trich o c y s t s are able to discharge). An important point made by the authors i s that trichocyst attachment apparently brings 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 point further, suggesting that trichocyst attachment may t r i g g e r other changes i n membrane organization, and that these attachment-dependent changes may be important f o r membrane-nucleus- int e r a c t i o n s . This would explain the t i g h t coupling between trichocyst attachment and nuclear behaviour at c e l l d i v i s i o n . 128 Recent experimental evidence suggests that the lesions due to the tam mutations may be d i r e c t l y a f f e c t i n g the trichocysts themselves, rather than the sequence of migration and attachment of the tr i c h o c y s t s . Evidence f o r t h i s came from a study employing microinjection of cytoplasm containing 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 (football-shaped tr i c h o c y s t s that w i l l not discharge) as the host c e l l and injected cytoplasm containing motile, wild type t r i c h o c y s t s . He found that within ©ne hour the wild type trichocysts had aligned and attached, and were capable of discharge, i n the foreign cytoplasm. However, i n j e c t i o n of cytoplasm containing 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 , revealed that these tric h o c y s t s remained nonmotile and incapable of discharge. Wild type trichocysts introduced into nd A or tam 8 host c e l l s remained motile and were capable of discharge. This study demonstrates that the mutations, nd A and tam 8, appear to d i r e c t l y a f f e c t the trichocyst i t s e l f . This would agree to a certa i n extent with the p o s s i b i l i t y of a membrane disorder being the common l i n k i n the p l e i o t r o p i c effect of the tam gene. Changes i n the permeability of c e l l membranes (or organelle membranes) can re s u l t i n size and shape changes 129 . (see Kasturi Bai 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. These l a t t e r changes w i l l i n t e r f e r e with microtubule (microfilament) interactions and possibly organelle movement. Thus, a l l the experimental evidence to date i s consistent with the p o s s i b i l i t y that 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 c e l l s . A phenomenon evident i n both tam G and tam 38 i s the formation of extra micronuclei and macronuclear anlagen at nuclear reorganization, a phenomenon not observed i n tam A. This s i m i l a r i t y i n phenotype between tam G and tam 38 i s surprising since tam G has stubby t r i c h o c y s t s while both tam 38 and tam A have football-shaped t r i c h o c y s t s . The possible existence of nucleus-cortical attachments during d i f f e r e n t i a t i o n of the synkaryon d i v i s i o n products remains unexplored. However, Sonneborn (1954) c l e a r l y demonstrated that the synkaryon products move to a s p e c i f i c region of the reorganizing c e l l , i n order to undergo normal d i f f e r e n t i a t i o n . This movement towards s p e c i f i c cytoplasmic regions (anterior and posterior poles of the reorganizing c e l l ) probably involves guidance from microtubules, and t h i s guidance may require nucleus- i c o r t i c a l membrane linkage. It i s possible that when the ,130 synkaryon comes into a s p e c i f i c area 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 of tam G, then, improper s p a t i a l positioning of the synkaryon may lead to inaccuracies 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 products. 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(1967) Structural aspects of amitosis: A l i g h t an electron microscope study of.the i s o l a t e d macronuclei of Paramecium a u r e l i a and Tetrahymena pyriformis. Chromosoma vol:23 , 59-79-141 Woodard, J. , B..Gelber and H. Swift (1961) Nucleoprotein changes during the mitotic cycle i n Paramecium a u r e l i a . / E x p t l . C e l l Res, vol:2 3 , 2 5 8 - 2 6 1 . 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. C e l l  Tissue Kinet. vol: 9 . 1 1 9 - 1 3 0 . Young,.E.G., and R.B. Cambell (1947) The reaction of beta-beta'-dichlorodiethyl sulphide with proteins. 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 routinely i n mutagen experiments involving Paramecium (see Sonneborn, 1975). McKay and Wright (194-7) were the f i r s t to synthesize and analyse NTG. The cytotoxic, carcinogenic, mutagenic and chemotherapeutic e f f e c t s have generated considerable interest i n t h i s monofunctional a l k y l a t i n g agent,. Since 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 from t h e i r i n t e r a c t i o n on genetic material, i t was proposed that DNA was the most l i k e l y target. Young and Cambell (1947) were amoung the f i r s t to report the sus c e p t a b i l i t y of the guanine moiety to attack by a l k y l a t i n g agents. Since then, a number of authors have presented evidence that the N-7 atom of guanine i s the most reactive s i t e . Lawley and Wallick (1957) demonstrated that the N-7 atom of guanine i s p r e f e r e n t i a l l y alkylated (support f o r t h i s f i nding came from a r t i c l e s by Reiner and Zamenhof [1957] and Alexander, et a l . , [1961]). These r e s u l t s agree with 142 143 the wave-mechanical studies of Pullman.and Pullman (1959) who. showed that the..N-7 atom of guanine was the most nucleophilic s i t e i n both the unpaired 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 . The Watson-Crick model for. the structure of DNA i s also consistent with this.observation, and shows that the N-3 s i t e of adenine and the N-7 s i t e of guanine are the most accessible of the possible reactive s i t e s . By using 14C-labelled NTG and 14 C-methyl methane-sulphonate (MMS), Lawley and Brooks (1963) showed that the major products from treated, undenatured DNA were 7-methyl guanine and 3-methyl adenine. Schoental and Bensted (1964) l a t e r showed that t h i s was also true f o r other a l k y l a t i n g agents. Using l a b e l l e d methyl-nitroso-urethane, Schoental 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 vivo and i n v i t r o . Singer, et a l . (1968) found that the N-7 atom of guanine was the primary target i n double-stranded (ds-) , high molecular weight molecules of DNA, but they found that t h i s was not the case i n guanine containing molecules of low molecular weight, such as Tobacco mosaic virus RNA (TMV-RNA). Under conditions 1 4 4 favoring the guanine reaction (neutral, aqueous solution) 7-methyl guanine was the principle-product. However, under d i f f e r e n t conditions ( 6 5 % dimethylformanide) methylated cytosine was the major product, and the mutagenic effect of NTG was greatly enhanced. They also showed that the guanine reaction was suppressed within the vir u s , but, that the cytosine reaction was not, and that, i n vivo TMV-RNA, when treated with NTG resulted i n the same reaction product obtained from p o l y c y t i d i l i c acid i n v i t r o . A l k y l a t i o n of guanine can lead to several d i f f e r e n t types of aberrati ons. A l k y l a t i o n of the N—7 atom of guanine, causing i o n i z a t i o n of the guanine moiety, can lead to mispalring of the ionized guanine to thymine instead of to cytosine (Auerbach, 1 9 7 6 ) , which would r e s u l t i n a t r a n s i t i o n ffrom G-C to A-T a f t e r DNA r e p l i c a t i o n . Both t r a n s i t i o n s (purine replacing purine or pyrimidine replacing pyrimidine) and transversions (purine replacing pyrimidine or pyrimidine replacing purine) appear to be the most common eff e c t s of NTG mutagenesis i n eucaryotes. i Another source of error i s the d e s t a b i l i z i n g effect of a l k y l a t i o n on the glycosi d i c bond (the bond between the sugar-phosphate backbone of DNA and the base). . _ \ 145 Lawley and Brooks (1963) have found that at neutral pH there is.a_ slow leaching out of the 7-alkylguanines and ' 3-alkyladenines from the DNA giving r i s e to apurinic gaps. These gaps may lead to t r a n s i t i o n s or transversions i f f i l l e d i n c o r r e c t l y or they maye lead to nonsense codons i n RNA, a f t e r t r a n s c r i p t i o n , giving r i s e to premature termination of t r a n s l a t i o n . The leaching out may also lead to deletions, giving r i s e to frameshift type mutations, or they may lead to f i s s i o n of the chain and eventually i n double-stranded f i s s i o n . H. Mailing and De Serres (1970) studied the genetic a l t e r a t i o n s of NTG mutagenesis on g e n e t i c a l l y marked heterokaryons of Neurospora crassa. Detectable a l t e r a t i o n s included point mutations, multilocus chromosome deletions and recessive lethalrrnidations i n the whole genome. Complementation analysis of NTG induced ad-§B mutants showed that there was a high frequency of G-^ C base pairs at the s i t e of mutation and that the major ef f e c t of NTG was inducing base p a i r substitutions from G-C to A-T. Although G-C to A-T t r a n s i t i o n s were found to be most common i n organisms with double-stranded DNA , t h i s was not found to be the case i n organisms with single-stranded DNA. Baker and Tessman (1968) found that G-C to A-T t r a n s i t i o n s occurred i n equal proportions i n S 13 phage, as against G-C to A-T 146 s p e c i f i c t r a n s i t i o n s i n Bacteriophage. McCalla (1965) found that NTG was. an e f f e c t i v e bleaching agent, i n h i b i t i n g the production of chloroplasts i n Euglena g r a c i l i s . The chloroplast DNA i s very r i c h i n adenine and thymine, and he suggests that only those agents which exert t h e i r mutagenic effect on A-T base pairs could be e f f e c t i v e bleaching agents. So, i n Euglena chloroplast DNA i t would seem that adenine i s the reactive target rather,-than guanine. Most of the NTG mutations ari s e as primary lesions i n the DNA, that are converted to f i n a l mutations at DNA r e p l i c a t i o n . There i s current evidence that NTG may act at or very near the r e p l i c a t i o n fork, and that NTG induced mutations ari s e only at the s i t e of r e p l i c a t i o n . Auerbach (1976) suggests that t h i s apparent e f f e c t i s due to a greater a c c e s s i b i l i t y of the bases to the a l k y l a t i n g agentat regions where there i s strand separation. Cerda-Olmedo (1976) suggests that NTG a c t u a l l y i n t e r a c t s with the DNA polymerase, causing the enzyme to act as a pheno-t y p i c a l l y mutagenic polymerase, which makes errors at r e p l i c a t i o n by incorporating the wrong base. The p o s s i b i l i t y of direct action on the enzyme came from studies that showed s p e c i f i c enzymes, such as beta-galactosidase, were 1 147 very sensitive to treatment with NTG and were i n fact inactivated i n the presence of NTG (Cerda-Olmedo, 1976) . [ I f the enzymes were inactivated by NTG treatment how could they incorporate the wrong base?]. The author also found that NTG e f f e c t s protein synthesis by inducing f a u l t y t r a n s l a t i o n . Studies by Nestman (1975) on continually growing cultures of Escherichia c o l i treated with NTG or EMS (ethyl methane sulfonate) showed that f o r NTG mutagenesis with the mutator gene (mut H) that the mutagenic ef f e c t was growth-rate dependent. Comparison of NTG mutagenesis on mut H and wild-type strains (mut +), show that the increase i n the rate of mutation i n mut H over mut + i s much greater than could be explained by an additive e f f e c t . The synergistic effect of NTG on mut + i s a 48 f o l d increase i n the rate of mutation. The synergistic responses of chemical mutagens i n t e r a c t i n g with mutator genes have apparently been interpreted as involvement of the mutator gene product with DNA r e p l i c a t i o n (Nestman, 1975) . The accumulating evidence (Lee and Jones, 1973; Cerda-Olmedo, e i a l . , 1968; Schimmer and Loppes, 1975) suggesting that NTG acts at the DNA r e p l i c a t i o n fork can be interpreted i n another manner. The alt e r n a t i v e 148 i s that alkylated lesions In DNA are ra p i d l y repaired except at the point of DNA r e p l i c a t i o n . Thus the only mutations that become f i x e d are those occurring i n the, region of the r e p l i c a t i o n fork. This i n t e r p r e t a t i o n agrees with the evidence of NTG damage repair i n P. t e t r a u r e l i a (Kimball, 1970). Kimball (1970) showed that when there i s a long lag period between treatment with the mutagen (NTG) and r e p l i c a t i o n of the genome the mutation rate approaches zero. Since most of the damage caused by NTG ( t r a n s i t i o n s , transverslon, etc.) i s repairable, Kimball's r e s u l t s suggest that the primary lesions are simply being repaired before they become f i x e d at anywhere along the DNA except at or near the region of r e p l i c a t i o n . APPENDIX II SPOT, l i n e 40-4b 2 A. Generation time: The mean generation time of homozygous sp_ ^ cells was 5.20-.08[S.E.] hours compared to 6.92±.27[S.E.] hours for the double mutant tam G/tam G;sp/sp and 5 . 0 0 i.ll[S.E.] hours f o r wild type c e l l s . 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 or micronuclear missegregation. I n t e r f i s s i o n c e l l s frequently contained one or more large, black, c i r c u l a r cytoplasmic inclusions (Plate 13, A and B). Position of these inclusions varied, but i n the majority of c e l l s they were a n t e r i o r l y placed. The median number of inclusions per c e l l was one, but occasionally up to three inclusions per c e l l were observed. High power (x 1000) observations on fi x e d , unstained c e l l s , revealed that the dark inclusions 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 v e s i c l e . 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 to the u r i c acid c r y s t a l s found scattered through out the cytoplasm of wild-type c e l l s . Apart from the 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 acid c r y s t a l s were present i n the c e l l ' s cytoplasm. In t h i s respect sji resembles the c_2 mutants described i n Sonneborn (1975). Penetrance of, the phenotype varied between clones and ranged from as low as o n e - i n - f i f t y to as high as eight-in-ten c e l l s . Observations on i s o l a t e d c e l l s containing one or more inclusions, revealed that i n cases where there was only one i n c l u s i o n that was anterior (majority of cases) t h i s i n c l u s i o n was retained by the proter at f i s s i o n . In cases where two inclusions were present, depending on the p o s i t i o n of the inclusions, both may be retained by the proter or they may segregate, one going to each s i s t e r c e l l . The C. Genetics: The s_p. t r a i t i s the consequence of a single, recessive mutation. The inclusions disappear within 24.00 hours afte r conjugation with wild-type c e l l s . The phenotype 151 reappears between f i v e to eight f i s s i o n s a f t e r autogamous segregation of the heterozygotes. This 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 to observe. Conjugants can be i d e n t i f i e d during p a i r i n g and immediately a f t e r separation of the conjugants. APPENDIX III THE ORIGINAL SELECTION SYSTEM The selection system used to recover the variant l i n e s was the second choice of two possible selection systems. The f i r s t choice, designed to select f o r spontaneous MR mutants, was a more complex system, designed by Berger (per. comm.) using a heterokaryon as the t o o l f o r selection (figure 20). Homozygous pw A c e l l s were crossed to homozygous t s l 111 c e l l s . The conjugants were subjected to heat shock, t h i s induced regeneration of the macronuclear fragments i n conjugants. Exconjugant clones exhibiting the pw A phenotype, were selected by the Dryl's method. A sample of c e l l s from each of the pw A clones was ra p i d l y expanded and put through autogamy. The genotype was then known. C e l l s , homozygous f o r pw A i n the macronucleus and heterozygous,for t s l / t s l + ; p w A/pw A + i n the micronuclei were backcrossed to homozygous t s l 111 c e l l s . Again, MR was induced by heat shocking the conjugants. 152 Figure 21. Formation of the pw A/pw A ; t s l + / t s l + (macronucleus) t s l / t s l + ; p w A+/pw A + ^micronucleus) heterokaryon. ma, macronucleus; mi, micronuclei MUTAGENESIS FIGURE 21 _ . 155 Phenotypically pw A exconjugant clones were selected f o r . The genotype of these clones was analysed by the same method mentioned before. C e l l s homozygous f o r pw A i n the macronucleus and either t s l / t s l ; p w A/^ pw A + or t s l + / t s l ; pw A+/pw A + i n the micronuclei were selected and expanded i n preparation f o r treatment with NTG. The heterokaryon has. two important values: f i r s t l y , a l l the c e l l s undergoing normal reorganization at autogamy, following mutagenesis, w i l l have macronuclei homozygous f o r the temperature sensitive l e t h a l s . These c e l l s could be k i l l e d by r a i s i n g the entire population of mutagen treated exautogamonts to the r e s t r i c t i v e temperature f o r 24.00 hours. The surviving c e l l s would be highly enriched f o r t s l + / t s l + ( i n the macronucleus) which either arose through MR or which were the progeny of c e l l s which did not undergo autogamy. Thus, putative, spontaneous MR mutants could be recovered e a s i l y by use of the JDW column (Kung, 1971). A number of problems were encountered i n synthesizing the heterokaryon and i n maintaining the c e l l s through to mutagenesis. The major problem encountered was preventing the c e l l s from going into autogamy, and thereby reverting to t h e i r o r i g i n a l genotypes. The process of building the new genotype took many c e l l generations, and even i f the c e l l s were well fed, the old c e l l s have a strong tendency to enter autogamy. Normally c e l l rejuvination takes place . . 156 at conjugation, but when macronuclear regeneration i s induced, . the c e l l s r e t a i n the parental macronucleus and therefore the parental age. The f i r s t mating i s conducted between very young clones, but many generations are used up by having to analyse the genotypes a f t e r each cross, and i n expanding the c e l l s f o r mutagenesis. Although the heterokayon was completed on a few occasions, c e l l s always entered autogamy p r i o r to mutagenesis. It was for. t h i s reason that the selection system was f i n a l l y abandoned. Other problems encountered with t h i s system included synchronization of mating pairs and the induction of MR by heat shock. The pw A/pw A c e l l s and the t s l / t s l c e l l s grow at very d i f f e r e n t rates and therefore cultures have to be set up with d i f f e r e n t amounts of food i n the cultures. These cultures are then cross-matched u n t i l mating reactive c e l l s from both l i n e s are obtained. A series of minor experiments were conducted to esta b l i s h the best combination of variables i n the induction of MR by heat shock. Synchronized mating pairs were selected between 5 - 5 i hours a f t e r p a i r formation and raised to the - r e s t r i c t i v e temperature f o r varying lengths of time. The 157 exconjugants were then r e i s o l a t e d and allowed to 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 regenerating fragments. The best combination of variables was a temperature of 34.5-34»7°C f o r a period of f i v e hours (Table XVII). Below 34.5°C MR induction was minimal and above 34.7°C was l e t h a l to the conjugants. The age of the conjugants was choosen to coincide, with 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. # PAIRS TEMP °C LENGTH SHOCK # 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 " 37.2 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 — Conjugants were 5.00-5.25 hours old when they were raised to 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 translated as the absence of forming threads. B a s i c a l l y , i t i s nuclear d i v i s i o n without the condensation of chromosomes and the formation of a t y p i c a l mitotic apparatus. One of the e a r l i e s t references ( i n English) to amitosis i s that of Child (1900) when he was studying the reproductive organs i n the cestode Monieza expansa and noticed a d i s t i n c t absence of mitotic figures i n the prog l o t t i d s , where the reproductive organs were d i f f e r e n t i a t i n g . He confirmed the observations, on the occurrance of amitotic nuclear d i v i s i o n i n the prog l o t t i d s , on several other occasions ( Child, 1902, 1904, 1907, 1910 and 1911). Nathanson (1900) found that amitosis could be induced i n Spirogyra by adding a few drops of ether to the water. This induction of amitosis was completely r e v e r s i b l e by retransferringjthe Spirogyra to fresh water. Jordon (1913) recorded the exclusive amitotic nuclear d i v i s i o n i n the epididymus of the white mouse. He stated that while studying 159 160 spermatogenesis i n the white mouse, there was not a single mitotic figure i n evidence, although many nuclei were i n varying stages of amitosis. There were many other references to amitosis i n various tissues that underwent rapid growth or that were i n starved conditions (Holmes 1914; Lynch, 1921, f o r example). More recently, Kocherezhkina and Korobko (1974) observed amitosis i n the testes of. several species of Black Sea crab, i n areas of rapid c e l l p r o l i f e r a t i o n . The authors suggested that amitosis i s a phenomenon i n i t ' s own right that has arisen i n response to an overwhelming increase i n the amount of nuclear material 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 contains between 800-900 haploid equivalents of DNA (Woodard, et a l . , 1961; A l l e n and Gibson, 1972; Berger, 1973; Morton, 1974), i t would seem reasonable to agree with t h i s idea. / 161 Plate 1. Various aspects of macronuclear misplacement i n dividing tam A homozygotes. A. Normal placement and elongation of the macronucleus. B. Normal placement and p a r t i a l elongation of the macronucleus. C. P a r t i a l elongation of the macronucleus and s l i g h t misplacement of the macronucleus toward the proter. D. Rarely observed misplacement of the macronucleus toward the opisthe". E. No macronuclear elongation and complete retention of the macronucleus i n the proter. F. P a r t i a l elongation and complete retention of the macronucleus i n the proter. G. Complete macronuclear missegregation to the proter and a $-1 abnormal micronuclear d i s t r i b u t i o n . H. Double macronucleate c e l l showing elongation of one macronucleus and no elongation i n the misplaced macronucleus. I. Segregation without d i v i s i o n of two macronuclei. 163 Plate 2. A comparison of complete and p a r t i a l macro-nuclear missegregation. A. Complete macronuclear missegregation. • B, Extreme p a r t i a l macronuclear missegregation. C, Slight p a r t i a l macronuclear missegregation. D. Equal, amitotic d i v i s i o n of the macronucleus. 16$ Plate 3. Vegetative tam A, and tam G homozygotes. A. Complete missegregation of the undivided macronucleus i n tam A/tam A c e l l s . Notice the six micronuclei aligned along the l i n e of f i s s i o n . B. Amicronucleate tam A/tam A c e l l . C. Amacronucleate c e l l apparently mating with a normal macronucleate individual,. The amacronucleate c e l l i s tam G/tam G crossed to pw A/pw A. P..Amicronucleate d i v i d i n g c e l l undergoing macronuclear missegregation. / 167 Plate 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 , giving 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 . A-D. Progressive stages of c e l l d i v i s i o n showing the formation of a vegetative macronuclear fragment (arrows). 168 Plate 5. Amacronucleate vegetative c e l l s of tam A homozygotes showing i r r e g u l a r numbers of micronuclei. A. Amicronucleate B. Amacronucleate C. Amacronucleate D. Amacronucleate E. Amacronucleate F. Amacronucleate amacronucleate c e l l , c e l l with 1 micronucleus. c e l l with 2 micronuclei. c e l l with 3 micronuclei. c e l l with 5 micronuclei. c e l l with 7 micronuclei. V 171 Plate 6 . Vegetative phase of the l i f e cycle showing i r r e g u l a r d i s t r i b u t i o n s of micronuclei 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 micronuclei. B. Irregular divider showing a 2-0 d i s t r i b u t i o n of micronuclei. C. Irregular divider showing a 3-3 d i s t r i b u t i o n of micronuclei. , D. Irregular divider showing a 4-4 d i s t r i b u t i o n of micronuclei. E. Irregular divider showing a 5 - 5 d i s t r i b u t i o n of micronuclei. F. Irregular divider 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 divider showing a 6 -6 d i s t r i b u t i o n of micronuclei. 173 Plate 7. Exautogamous tam A/tam A c e l l s just completing the f i r s t exautogamous c e l l 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 missegregation (C) of the posterior macro-nuclear anlagen (ma). D and E. S i s t e r c e l l s of exautogamous tam A homo-zygoteshaving undergone missegregation of the macronuclear anlagen. Notice how the majority of fragments passed to the other s i s t e r c e l l . F. Normal segregation 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 Plate 8. Exautogamous tam G/tam G c e l l s just completing the f i r s t postautogamous c e l l cycle. A. Exautogamous c e l l showing early evidence of macronuclear regeneration i n the presence of one underdeveloped anlage. B. Normal segregation of 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 prezygotic macronucleus are begining to show signs of regeneration. C. Irregular exautogamont with 4 macronuclear anlagen. D. Irregular exautogamont with 7 macronuclear anlagen. '177 Plate 9. The completion of the f i r s t c e l l cycle following autogamy i n tamJl/tam_A c e l l s . A. Normal segregation of the macronuclear anlagen. B. Grossly underdeveloped macronuclear anlagen (ma) and the fragments were undergoing regeneration. C. Improper placement of one macronuclear anlage (ma), lack of suppression of DNA synthesis i n the fragments (fg s ) . D. Proper alignment of regenerating macronuclear fragments ( f g s ) . E. Unequal segregation of the remaining macronuclear fragments (fgs) at c e l l d i v i s i o n . F. Macronuclear fragments are of grossly unequal size and the divider 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 micronuclei (mi). (7$ 179 Plate 10. Exautogamous c e l l s from l i n e 21-3c. A. Amicronucleate exautogamont showing grossly unequal development of the macronuclear anlagen. B. Amicronucleate exautogamont without macronuclear anlagen and with nonregenerating prezygotic macronuclear fragments. C. Multiple micronuclei i n an exautogamont without macronuclear anlagen. The prezygotic macronuclear fragments appear shattered and undergoing a u t o l y s i s . D. Very early evidence of regenerating macronuclear fragments i n the possible absence of micronuclei. Plate 11. Exautogamous c e l l s belonging to l i n e 21-3c. A. An amicronucleate c e l l showing lack of macronuclear regeneration i n the absence of the suppressive macronuclear anlagen. Notice the unusual, large 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 regeneration i s i n evidence and no food vacuoles are present. 183 Plate 12. 21-3c conjugants showing lack of nuclear 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 , only 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 pa i r i n g i n sexual union with pw A/pw A conjugant. D. A s i m i l a r conjugant pair as described f o r C , but notice the exchange of DNA across a cytoplasmic bridge. Plate 13. Cytoplasmic inclusions c h a r a c t e r i s t i c of sp_ homozygotes. ( 

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