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Selection system for temperature sensitive mutants in Paramecium aurelia Baumann, Paul John 1973

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A SELECTION 'SYSTEM FOR TEMPERATURESENSITIVE MUTANTS IN PARAMECIUM AURELIA by Paul John Baumann B.Sc, University of British Columbia, 1969  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in the Department of Genetics  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA November, 1973  In p r e s e n t i n g an the  this  thesis in partial  advanced degree at the Library  University  s h a l l make i t f r e e l y  f u l f i l m e n t of the  of B r i t i s h Columbia, I agree  a v a i l a b l e f o r r e f e r e n c e and  I f u r t h e r agree t h a t p e r m i s s i o n f o r extensive for  s c h o l a r l y p u r p o s e s may  by h i s r e p r e s e n t a t i v e s .  be  g r a n t e d by  thesis for financial  written  permission.  gain  Department The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a  the  Head o f my  Columbia  s h a l l not  be  that  thesis  Department  copying or  for  study.  copying of t h i s  I t i s understood that  of t h i s  requirements  or  publication  allowed without  my  (i) ABSTRACT Four temperature-sensitive DNA deficient mutants of Paramecium aurelia have been isolated through a new application of UV light photolysis of 5~bromouracil substituted DNA, following nitrosoguanidine mutagenesis!  Bromouracil was introduced by bacterial vector to achieve  specific incorporation into DNA.  Non mutant cells presumably incorporated  5-bromouracil at the restrictive temperature, mutants did not.  The  selection procedure took a month to complete, requiring two successive sexual generations. There were two lethal periods for wild type cells i n the screening system; the f i r s t occurs during the irradiation of BU substituted macronuclei by UV light, the second, following autogamy (a result of homozygosis of UV light damage to the bromouracil labelled . micronuclei). From 1300 isolated survivors from the screening procedure, 29 lines were classed as temperature-sensitive.  Approximately Ik percent of these  were ts DNA deficient (the ratio of restrictive to permissive temperature incorporation was 10 percent or less).  Protein synthesis, and food-vacuole  formation at the restrictive temperature deviated no more than 50 percent from the normal level.  .  The selection procedure was shown to be efficient in retrieving ts DNA deficient mutants.  A genetically marked ts DNA  mutant (isolated  through the selection system earlier) was recovered at an enrichment of 7500 fold over the wild type (mean seven runs). The methods of production of bromouracil substituted bacteria have been standardized, and the durations of various parameters of the selection system have been set. For example, the length of UV light exposure following bromouracil substitution, and the length of restrictive temperature pretreatment  (ii) (Equilibration time before bromouracil treatment) have been optimized. The relative effects of parts of the screening procedure are presented along with discussion of the r e s u l t s , and suggestions f o r future a p p l i cations of the isolated Paramecium l i n e s .  (iii) TABLE OF CONTENTS PAGE L i s t o f Tables . . . . . . . . . . . . . . .  . .  L i s t o f Figures Acknowledgement  iv v  . . . . . . . . . . . . . . . . . . . .  •  vi  Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . .  1  M a t e r i a l s and Methods  5  . . . . . . . . . . . . . . .  Results I  Y i e l d o f the S e l e c t i o n System on Mutagenized Populations  . . . . . . . . . . . . .  15  . I I E f f i c i e n c y o f the S e l e c t i o n System (Marked-Recapture Experiment) III IV  . . . . . . . . . . . . . . .  25  Pattern o f Decrease i n S u r v i v a l and Clone Forming A b i l i t y Due t o S e l e c t i o n . . . . .  26  R e l a t i v e Contribution o f Components o f the S e l e c t i o n System t o T o t a l C e l l Death . . . . . . . . . . . .  28  Discussion Mutagenesis and the S e l e c t i o n System  .....  ...  32  . . . . . . . . . . . . . . . . . . .  37  . . . . . . . . . . . . . . . . . . . . .  39  Rationale f o r the S e l e c t i o n System and Possible Applications Concluding Statement Notes  .  Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . .  40 43  Appendices I II  Growth Curves f o r E s c h e r i c h i a c o l i Used . . . . . . . . .  o . .  T h e o r e t i c a l Scheme f o r the Use o f ts'DNA D e f i c i e n t Mutants i n an Experiment Concerning the Control o f DNA Metabolism . . . . . . . . . .  49  52  (iv) LIST OF TABLES PAGE I II  III  Isotopes Used . . . . . . . . . .  •  Incorporation of DNA Precursors from Labelled Bacteria into Paramecium aurelia at 23 and 35°C  9  Fate of Mass Cultures During the Mutagenesis Treatments . • • . • • • • • • * • • » . « . . .  IV  7  H 15  Yield of the Selection System  V• Putative Mutant Group Analysis a  Incorporation of DNA Precursor at 27 and 37°C Part 1  Quantitative -^H-Thymidine Incorporation . . . . . . . . . . . . . . . . . .  19  3  Part 2 Part 3  Approximate ^H-Thymine Incorporation . . . . . . . . Quantitative ^H-Thymine  ....  20  Incorporation . . . . . . . . . . . . . . . . . .  21  b  Protein Synthesis at 27 and 37°C  c  Food-vacuole Formation at 23 and 35°C  d Growth at 27°C . . . . . . . VI Efficiency of the Selection System (Marked-Recapture Experiment) VII  Relative Contribution of Components of the Selection System to Total Cell Death .  22 . . . . . . . . . . .  23  . . . . . . . . .  24 25 29  (v) LIST OF FIGURES PAGE 1  Selection System . . . . . . . .  . . . . . . . . .  4  2  5-Bromouracil Incorporation into Bacteria  . . . . . . . . . .  8  3  Bacterial Colony Formers Relation to Turbidity . . . . . . . .  8  4  A  Vegetative Survival as a Function of Exposure to UV Light . . . . . . . . . . . . . . . . Clone Survival with Varying Light Exposure . . . . . . . .  2? 27  Effect of Light Exposure and Delay Before Isolation on Clone Survival . . . . . . . . . . . . . . . .  31  B 5  (vi) ACKNOWLEDGMENT  I wish t o thank Dr. J . D. Berger f o r h i s constant advice and reassurance without which t h i s work could not have been completed.  A l s o , I thank Robert Hunter, Glenn  Morton, and E r i c Peterson f o r t h e i r good humour i n the face of temporary d i s a s t e r and f a i l u r e .  The t e c h n i c a l  a s s i s t a n c e and c o n s t r u c t i v e c r i t i c i s m given by E r i c was e s p e c i a l l y appreciated. This research was supported by N.R.C. operating grant A-63OO t o Dr. J . D. Berger.  INTRODUCTION C o n d i t i o n a l mutations provide one genetic means t o examine e s s e n t i a l f u n c t i o n s i n any prokaryotic o r eukaryotic organism. The Bar locus i n Drosophila melanogaster was the f i r s t gene shown t o have v a r i a b l e expression as a f u n c t i o n o f temperature (Seyster, 1919; D r i v e r , 1931)• However, c o n d i t i o n a l mutations o f t r u l y i n d i s p e n s i b l e functions were not e x p l o i t e d u n t i l S t r e i s i n g e r e t a l . . (1961) i s o l a t e d temperature s e n s i t i v e ( t s ) mutants o f lysozyme i n E s c h e r i c h i a c o l i . E p s t e i n et a l . (19&3) ^ d Edgar and L i e l a u s i s (1964) used t s mutations of bacteriophage T^ to study c o n t r o l s o f e s s e n t i a l metabolic and deveXopsental processes.  Since that time t s mutations have turned out  to b e an i n c r e a s i n g l y important genetic t o o l .  Ts mutants have been  used t o study macromolecular synthesis i n E s c h e r i c h i a c o l i 1964) ao£ i n Saccharomyces c e r e v i s i a e ( H a r t w e l l , 1967).  (Neidhart,  This c l a s s o f  mutations', has a l s o been used to probe development i n D i c t y o s t e l i u m discoideam (Loomis, 1969) ajid i n Drosophila melanogaster (Suzuki, 1970). WA synthesis i s an e s s e n t i a l f u n c t i o n which has been vigorously explored g e n e t i c a l l y through use o f c o n d i t i o n a l mutants i n prokaryotes ( K l e i n and Bonhoeffer, 1972; Gross, 1972).  However, f a r  l e s s work has been done on the genetic b a s i s o f DNA r e p l i c a t i o n i n eukaocyotes ( H a r t w e l l , 1967; Unrau and H o l l i d a y , 1970).  Such studies  have Jbeeft hampered by the l a c k o f a p r e l i m i n a r y screening procedure which could be a p p l i e d to mass c u l t u r e s ; most o f the mutants have been recovered! b y r e p l i c a - p l a t i n g o f i s o l a t e s a t the permissive and r e s t r i c t i v e temperatures.  The goal o f t h i s study was t o devise a  preliminary screening procedure, a p p l i c a b l e t o mass c u l t u r e s o f Paraiaeciga a n r e l i a . which would allow s e l e c t i o n o f t s DNA d e f i c i e n t  2 mutants. The screening procedure i s based on 313 nm. ultraviolet (UV) light photolysis of 5""taromouracil (BU) substituted DNA.  Thymine dimer formation  has long been recognized as a result of 275 nm. UV light irradiation of DNA (Wacker, Dellweg, and Weinblum, 196l).  Unlike shorter wavelengths,  313 nm, l i g h t has l i t t l e or no effect on DNA unless i t i s BU•substituted (Dodson, Hewitt, and Mandel, 1972). label A  Using BU and radioactive phosphate to  phage DNA, they demonstrated a linear release of phosphate in BU  substituted cultures and no release i n non-BU substituted controls with duration of UV exposure.  UV photolysis (313 nm.) of BU labelled DNA  produces single strand breaks due to debromination of BU i n the DNA, subsequent free radical formation, and consequent breakage of the DNA at the 5 ' nearest deoxyribose neighbour (Dodson, Hewitt, and Mandel,  1972}  Benhur and Elkind, 1972). To take advantage of this increase i n UV sensitivity, bacteria containing BU labelled DNA were fed to the paramecia.  Animals that  synthesized DNA at the restrictive temperature, incorporated BU from the bacteria into their genomes and were selectively k i l l e d by exposure to 313 nm. l i g h t emitted by flourescent tubes (Ley and Setlow, 1972), while cells that did not synthesize DNA at the restrictive temperature were spared. The idea was suggested by J . D. Berger and was based on the work of Puck and Kao (1967) who devised a screen for nutritional mutants i n tissue culture cells using BU photolysis. . UV photolysis of BU substituted DNA (Boyce and Setlow, 1963)  has  been used i n radiation sensitivity assays i n Escherichia c o l i (Greer and Zamenhof, 1957)  and bacteriophage T^ (Stahl et a l . , 1961).  It has also  been used, i n Escherichia c o l i to recover ligase mutants (Pauling and Hamm, 1968) and i n Chinese hamster cells to obtain purine requiring (Taylor,  Souhrada, and McCall, 1971) and proline requiring mutants (Puck and Kao, 1967) as mentioned. The selection system as applied to Paramecium bulk cultures i s shown i n Figure 1.  Both vegetative death (position l ) and post-autogamous  death (position 2) are achieved routinely to produce greater than 99«9 percent k i l l i n g i n the selection runs.  The vegetative l e t h a l i t y was  not expected because the chance of damaging a l l 800 copies of a gene responsible f o r a particular essential function i n the macronucleus seemed small. This fortunate vegetative death made selection of large numbers of c e l l s possible; without i t , only r e l a t i v e l y small numbers of c e l l s could have been screened since individual c e l l l i n e s would have had to be followed through the following autogamy (position 2). Overall, when both types of death are summed, the Selection System enriches f o r a t s mutant \ obtained through the screen, by approximately 7500 f o l d over the wild type.  The occurrence of autogamy, and the ease of  mass culture handling are the major advantages of Paramecium as an experimental eukaryotic organism f o r studies of this type.  Autogamy, a  s e l f - f e r t i l i z a t i o n process which produced homozygosis of a l l l o c i i n a single step, allows recessive mutations to be expressed without an inbreeding cross. Limits and Goals of the Present Study I t was hoped that the project would produce a selection system f o r t s DNA deficient c e l l s that was p r a c t i c a l enough f o r use on a routine basis. These expectations have been f u l f i l l e d . This study has been limited to the d e f i n i t i o n of the screening procedure design and has l e f t the generation and analysis of large numbers of mutants to others.  A  figure 1 SELECTION  SYSTEM  F  E  E  D  Equilibration BACTERIA °y  35  6hrs +6hrs f 1hr  K  DARK  NG /17  27  o  t  \ \  Expression  Feed  Autogamy Autogamy  Test  position 2  IRRADIATE  1/  12 hrs position 1  Start t-  1 DAY  NG  Expres- Veg Exautogamous death ^ sion j j death + —I—I— + 22 24 27 7 8 11 1213 15 18 Auto- Selection Autogamy Testing gamy 35°C |  5 MATERIALS AND METHODS Growth and Handling of Paramecla Paramecium aurelia. syngen 4, stock 51» was grown on Cerophyl rye grass medium (Cerophyl Laboratories, Kansas City, Mo., U.S.A.) with Aerobacter aerogenes as the food organism (Sonneborn, 1970).  Mass cultures  were grown i n p l a s t i c boxes (30 x 22 x 10 cm.) holding approximately three to four l i t e r s of medium. Population densities of 1 to 3 x IO-' animals per ml. were routinely obtained.  The behavioural mutant pawn (Kung, 1971)  was  u t i l i z e d as a genetic marker i n the "Marked-recapture" experiments (section I I of Results). Survivors of the screening procedure were categorized f o r v i a b i l i t y according to Kimball (1963)»  "very poor" was equivalent to one f i s s i o n or  less i n the f i r s t three days following autogamy (unrestricted growth at 27°C)j "poor", two f i s s i o n s ; "bad", three f i s s i o n s ; and "good", four or more f i s s i o n s .  These groups were lumped into "subviable", zero to two  f i s s i o n s , and "viable", three o r more divisions per three days. 3 3 ^H-thymidine and •'H-leucine uptake was measured by counting the 2 number of s i l v e r grains (autoradiographic) i n a 50/J.  area and calculating  the ratio of incorporation at the r e s t r i c t i v e and permissive temperature, (Table Va, part l,and Table Vb).  In the second part of Table Va, a rough  estimate of t h i s r a t i o was made -using a 5 category ranking scale; then promising l i n e s were counted (lines 124 and 1 appeared to incorporate less at the non-permissive temperature, Table Va, part 3)»  Food-vacuole  formation was determined using d i r e c t count of vacuoles containing carbon p a r t i c l e paint after 20 to 30 minutes (Table Vc, part l ) .  Again i n part 2  of Table Vc, the incorporation into food-vacuoles was crudely determined on a ranking'scale with f i v e categories (heavy to no incorporation). In these  food-vacuole experiments, cells were fed water colour carbon paint (Giinther Wagner "Pelikan" #730) i n Cerophyl medium at both temperatures and fixed with 0.5 percent gluteraldehyde, affixed to albumenized slides and counted. Dippell's stain (Dippell, 1955) was used to identify macronuclei that were fragmented.  This was a test for nuclear reorganization which  occurs during conjugation or autogamy. Bacterial Strains and 5-bromouracil Substituted Bacteria Escherichia c o l i 15 TAU~ and 15T~ 55-7 were grown on Modified M-9 medium (Pierucci, 1969), supplemented as required with casamino acids (Difco), uracil,thymine, tryptophane, and thiamin. Growth curves f o r these strains can be found i n Appendix I. Production of BU substituted Escherichia c o l i was done routinely by inoculation of 750 ml* of thymine (lOyUg/ml.) supplemented medium from a 10 ml. overnight culture.  When an optical density of 0.05-0.1  was reached, the cultures were centrifuged and washed i n modified M-9 salts three times.  The pellet was resuspended i n medium supplemented with  5-bromouracil (25yW g/ml.) instead of thymine.  The culture entered  stationary phase at a turbidity of O.D. 0 , 3 - 0 . 4 after at least six hours of growth. The bacteria were stored at 4°C until a few hours before use^, when they were resuspended i n Dryl's salt solution (Dryl, 1959) ready to be fed to the paramecia.  TABLE I ISOTOPES USED Spec. Act. (c/mmol.)  Isotope  Source  3  H-thymidine (methyl-^H)  5.0  Amersham  ^H-thymine (methyl-^R")  1.9  Schwartz Mann  8.7  Schwartz Mann  •^H-5-bromouracil  (C6-\)  ^H-leucine (C4,5-^H)  31.9  New England Nuclear  Tritium labelled BU (Table i ) was used to measure uptake into 15T 55"7 Escherichia c o l i at 35°C. as the culture grew (Figure 2).  Label was taken up at an even rate  Bacteria were labelled from approximately  O.D. 0.08 with O.lyUC^/g of unlabelled base analog and permitted to grow for 240 minutes.  Samples were taken at 10 minute intervals and adhered to  Watman JWl cellulose and GF/A glass fiber f i l t e r discs (2.4cm. in diameter which had been presoaked i n a one percent solution of BU and a i r dried). The spotted f i l t e r s were immediately immersed i n five percent trichloroacetic acid with five percent BU and kept on ice for at least three hours.  Three successive washes i n the same but fresh solution  followed by alcohol-ethyl ether ( l s i ) and ethyl ether finished the precipitation.  The discs were then a i r dried at 80°C and placed into  liquid s c i n t i l l a t i o n vials, spot side up. material was then ready for counting.  The precipitated acid insoluble  Cell number was not estimated  directly by turbidity (optical density); but by clone forming a b i l i t y on  4 nutrient agar plates.  A plot of colony number versus optical density  (Figure 3) demonstrates the loss of exponential growth and entry into stationary phase i n both thymine and bromouracil supplemented cultures. If the bacteria should run short of DNA precursor before stationary phase  8 i s reached, the cells would exhibit the elongation ("chain" or "snake") effect well known i n Escherichia c o l i undergoing thymineless death or related metabolic crises (Cohen and Bamer, 195^} Menningman and Szybalski, 1962).  Cohen et a l . ,  1958;  In other words, Figure 2 i s based on  actual c e l l numbers (Figure 3 corrected) which cannot be directly estimated by turbidity.  The counting efficiency was higher with glass  f i l t r e s (GF/A) as shown by Pinter, Hamilton, and Miller (1963). FIGURE 2  FIGURE 3  5-BR0M0URACIL  INCORPORATION BACTERIA AT 36°C  BACTERIAL COLONY FORMATION VERSUS TURBIDITY  HGF/A  12-  /  §70-  r  'S8-  3MM  w1  "T 0  60  120 M I N U T E S  180  240  0  0.1 OPTICAL  0.2  0.3 DENSITY  Incorporation of DNA Precursor into Wild Type Paramecium using Bacterial Vector Incorporation of BU into Paramecium macronuclei was examined at 23°C and 35°C by allowing the cells to feed on labelled bacteria for six hours.  The cells were then washed three times i n unlabelled medium, dried  on albumenized slides, Feulgen stained, and autoradiographed.  The results  (Table II), show that incorporation i s not significantly lower at the  9 higher temperature.  The experiment indicated that BU entered the  macronucleus of cells i n a mass culture under selection system conditions. TABLE II INCORPORATION OF DNA PRECURSORS FROM LABELLED BACTERIA INTO PARAMECIUM AURELIA AT 23°C AND 35°C Label  Temp.  n*  Mean Grain Count ± SE  3,H-thymine  23°c 35°c  25 34  23.2 + 2.3 19.7 i 1.2  23°C  35 39  21.2 + 1.8  35°c  17.8 ± 1.4  0.66 0.62  *Sample sizes varied because some c e l l s were i n autogamy. Only cells with vegetative nuclei were scored. Liquid Scintillation Counting Liquid s c i n t i l l a t i o n counting was performed on an Isocap J00 Nuclear Chicago counter using 0.4^ PPO (2,5-Diphenyloxazole) and 0.01^ P0P0P (l,4-Bis-2-(5-phenyloxazolyl)-benzene) i n toluene as the fluor bearing cocktail. Preparation of Autoradiographs Labelled cells were washed by micropipette transfer through three successive depression slides containing non-labelled Cerophyl medium. The cells were then individually removed to albumenized slides, each i n a single microdrop and allowed to dry.  After fixation i n Carnoy's (ethanolacetic,  3«l) for 20 minutes, the slides were hydrolysed i n 1 Normal HC1 at 60°C for 10 minutes, and Feulgen stained f o r 60 minutes.  Liquid nuclear track  emulsion ( i l l f o r d K-5) was applied as a $Q>% w/w solution with water at 70°C. After the slides were dipped i n emulsion, they were dried i n an upright  10 position and then stored at 4°C i n l i g h t tight boxes.  The emulsion  was developed i n Kodak D-19; coverslips were mounted i n Permount. Whenever Escherichia c o l i was used as a vector f o r tritium l a b e l , i t was imperative that the nuclear a c t i v i t y be estimated autoradiograp h i c a l l y since both Paramecium DNA and bacterial DNA (caught i n the animals* food-vacuoles) would be counted by l i q u i d s c i n t i l l a t i o n techniques. Mutagenesis Paramecia were treated with N-methyl-N'-nitro-N-nitrosoguanidine (NG) according to Pollock (1970). The c e l l s were harvested by negative geotaxis (they congregate a t the neck of a four l i t e r erlenmeyer f l a s k , where they are easily siphoned o f f ) and gentle centrifugation.  These log  phase animals were then added to Dryl's solution and an equal volume of the mutagen i n Dryl's solution (to make a working solution with concentration, 75yUg/ml.)  60 minutes i n a protective glove-box.^ At  55 minutes the solution was centrifuged down, and the pellets were rescued at 58~60 minutes.  Three careful washes were performed before the animals  were returned to Cerophyl medium. Although Sonneborn (1970) used a nitrosoguanidine concentration of 20 to 50/Wg/ml. f o r 30 to 40 minutes he observed the same post-autogamous death as Pollock; i . e . , 40 to 60 percent. Exautogamous death, an index of damage to DNA i n a mutagenesis treatment, was determined by making individual isolations of animals following the chemical treatment along with untreated controls.  These clones were  followed u n t i l gene expression a f t e r autogamy when a ratio i n i t i a l number was calculated.  of dead to  To test exautogamous l e t h a l i t y , and to  check f o r losses of c e l l s during the treatment, extensive c e l l counts were made during two mutagenesis experiments (Table I I I ) .  In mutagenesis A,  11 the treatment caused a 9i4 percent decrease i n c e l l number. However i n Treatment B, the cells doubled i n number indicating that active metabolism TABLE III FATE OF MASS CULTURES DURING THE MUTAGENESIS TREATMENTS Mutagen Treat. A  Pretreat. Number  Post Treat. Number  4.0xl0  2.7x10^  -32.5^  2.6x10*  2.0x10*  -23. UK  1.4xl0  3.0x10"  114.3#  8.1x10*  138-.2J6  c  Exptl. Cont.  Loss* NG+ Handling  Loss NG 9.4^  c  3.4x10*  Cont.  occurred during the exposure to nitrosoguanidine. due to the toxicity of NG (23.9%)•  43.5#  40.US  3*1%  B  Exptl.  Post Autogamous Loss  23.9^  64.8?S 61.2% 3.6%  Again, there was a loss  In both experiments, dividing cells were  found when the culture was visually checked.  Exautogamous death of controls  was uniform at three to four percent but lethality i n the experimental cultures was 40 and 6 l percent respectively. Screening System Exautogamous cells that had been mutagenized were grown approximately 10 fissions to allow for phenotypic expression (phenotypic lag for most genes i s five to 10 fissions).  A sample of 5 x 10^ to 1 x 10^ cells was  heated to 3^«5°C for six hours i n a water bath and then BU substituted bacteria (prewarmed to 3^»5°C) were fed to the animals for six hours i n darkness.  This was followed by quickly but gently washing the cells by  centrifugation.  They were then resuspended i n Dryl's solution and placed  in petri dishes under the UV light.  The light source was a battery of  Westinghouse fluorescent tubes (F20 T12/CW) at less than three cm. distance.  12 The  c e l l s were p e r i o d i c a l l y checked f o r body d e f o r m a t i o n and l y s i s .  7  An  a d d i t i o n a l D r y l ' s wash s e v e r a l h o u r s i n t o t h e l i g h t exposure was done i f l a r g e amounts o f d e b r i s were p r e s e n t i n t h e c u l t u r e .  A f t e r 12 hours o f  l i g h t exposure a c t i v e l y swimming c e l l s were i s o l a t e d i n d i v i d u a l l y i n t o depression  s l i d e s w i t h c u l t u r e f l u i d and s t o r e d a t 17°C o r 27°C.  Animals  showing any s i g n s o f s w e l l i n g e a r l y i n t h e l i g h t exposure ( f o u r t o s i x h o u r s ) d i e d soon a f t e r i s o l a t i o n .  Consequently, t h e h e a l t h i e s t  looking  specimens were r e s c u e d f i r s t and t h e n a s m a l l amount o f c u l t u r e f l u i d  was  added so t h a t  rather  " l o s t " s u r v i v o r s had an o p p o r t u n i t y  t o f e e d on b a c t e r i a  t h a n the dismembered b o d i e s o f t h e i r l y s e d p e e r s . consume m a t e r i a l from l y s e d c e l l s .  Paramecia w i l l r e a d i l y  In s e l e c t i o n "runs, t h i s p r o b a b l y  i n c l u d e s f r e e BU l i b e r a t e d by l y s i s o f t h e m a j o r i t y ( i n c l u d i n g mutants) would t h e r e f o r e be v u l n e r a b l e r e s u l t o f t h i s secondary uptake.  of cells.  A l l cells  t o UV l i g h t damage as a  Thus t h e o p t i o n a l D r y l ' s wash was  often  u s e d ( t h e a n i m a l s were a l l o w e d t o swim up from t h e p e l l e t formed by c e n t r i f u g a t i o n , c o l l e c t e d and r e - e x p o s e d ) .  C r i t e r i a f o r Temperature S e n s i t i v i t y A l l c e l l s r e c o v e r e d from t h e s c r e e n i n g "survivors".  p r o c e d u r e were c a l l e d  Putative t s l i n e s that s a t i s f i e d a t l e a s t three  separate  t s t e s t s (zero t o one f i s s i o n a t t h e r e s t r i c t i v e temperature a f t e r 2M- h o u r s , and  t h r e e t o f i v e f i s s i o n s a t t h e p e r m i s s i v e temperature) were s e l e c t e d from  these s u r v i v o r s .  A t l e a s t two more t s t e s t s were conducted b e f o r e t h e l i n e s  were examined a u t o r a d i o g r a p h i c a l l y  f o r DNA  incorporation.  W i l d t y p e c e l l s r o u t i n e l y grew a t a r a t e o f f o u r t o f i v e f i s s i o n s p e r day  a t 27°C; a t 36°C, w i l d type c e l l s s u r v i v e d b u t d i d n o t d i v i d e .  35°C t h e c e l l s grew a t a meexi r a t e o f k.k3 Results).  A t 3^ t o  d i v i s i o n s p e r day (Table V o f  34.5°C was chosen t h e r e f o r e as t h e r e s t r i c t i v e temperature.  This  !3 i s i n disagreement with Sonneborn (1970) who states that 35 to excellent  i s "an  temperature".  At f i r s t , mutants were only retained i f they went four to five fissions per day at 27°C and one or less at the restrictive  temperature.  Subsequently, i t was reasoned that cells with less than 100 percent penetrance of the mutant phenotype, or with a slow-acting mutation (of many possible types) could conceivably make i t through a single period of DNA synthesis before halting prior to or during the next synthesis period. If an initiation mutant experienced a temperature shift during the macronuclear S period, i t would carry on u n t i l the next G^-S boundary before stopping.  Also, maintenance mutants might barely make i t through a  single synthesis. In the worst situation, one could imagine that both these phenotype-delaying  characteristics occurred together.  Since i t  takes greater than 200 minutes for an a i r incubator to heat the animals to the restrictive temperature, there i s reason for allowing a putative ts line i n a ts test to go two fissions at the restrictive temperature and s t i l l consider i t as a bona fide mutant. Ts tests are best performed i n a water bath, exposing individual dividers i n melting point capillary tubes to the restrictive  temperature.  The tests as performed had a delay before the true equilibrium of temperature was reached.  Preheating the slides helped somewhat. Despite these compli-  cations in the design of the tests, many lines indeed grew only one or less fissions per day at the restrictive temperature.  (Further remarks on  the t s tests are i n the Discussion^) Statistics on Putative Lines Standard errors were calculated on arithmetic means. However, the probability of equality of sample means between groups (P^) being one, was determined by computer from an analysis of variance on transformed data  14 (square root transformation) to minimize differences i n sample variances.  15 RESULTS I  Yield o f the Selection System on Mutagenized Populations A summary of a l l s e l e c t i o n runs i s shown i n Table IV.  The length  of the i r r a d i a t i o n exposure was varied considerably u n t i l approximately 12 hours was adopted exclusively (from experiment 7e on).  Of the f i v e  mutagenesis experiments, four were constructive; two yielded putative TABLE IV YIELD OF THE SELECTION SYSTEM I C O O P O O O O r H ^ t i - ) VT\vO XT\ I rH fe: CM rH rH  an  e  S-P «H tO © JQ  o  0  U  we  + + VO O N O O H ^ H O N N N V ^ ^ - J i-H CM UN C 0 H \ O » A ( M N CM rH CM rH  •rt cd rH -P to 00 H CO a o rH 3 cd CO £ © H|<W|<\>H|<\>H|<\J  N VO N 4 - C O CO CO 00 O N O N O \ O N O \ rH H rH CM  -3- • to rH o£  i-)|o)nf*-nW-inLfp.|4Ovj^ VT>  UN. \P» UN. UN UN VO UN UNM3 UN UN-3" XT) UN O O O O O O O O O O O O O O O  N  O  UN t>- C N O O CM -3" ON m o  CM ON CM 00 ON CO C ^ V O C\! \Q H  ^3"  4- ON C\! V O  ! !0 >0 S *0 "*> I  „  rH rH rH rH rH t>CNSCMC\l<NlC\JC\tvOvO\OvOvO  *  VO O „ O O O  CO • • ON rH  x>  <r -P  10  CD 4->  VO O  I  cd x» B J d ^ O T ) 0 ) d f l O ' d ( i ) UNVO o-o-c^o-o-ooooo  HHririH  •  rH XI CO o rH  O  CO CO xi © H -P vH cd O xi © rH c cd c CO c •v. X to s CO e  bp to U £ © -P ft <u-p © ft c X O © CO •P •rt •P 1 0 -p o O -H © o s © (0 ftCO rH 0) (1) C-P ©CO * 10 CO ! CO ? -H  -ch rH eg -P  s  rH  H U •  o to -p  >• rH O © c rH © M CH 3 e £> CO CO CO •p rH CO -p o cd © >o > xi u cd o t EH O to © -p  c  (0 XICO •p  ur  \ Q V O V O O - C~\ CM H O v O  xi a cd  ru  H|<M  O  CH  o  © *  © rH  O  o  rH  •rt  to  16 ts lines, (mutageneses number 5 and 10). The mutagenicity of these treatments (based on exautogamous death) varied from extremely high (95%)t to f a i r l y low (25%) • Forty to sixty percent exautogamous death was desired. The number of cells treated with nitrosoguanidine, and the number of cells selected, changed with each run.  6 animals (1x10  Between one and 20 million  6  to 2x10  being the most convenient) were treated i n a  single series of selections.  An optimum of about 5x10^ cells per  selection run became routine. Several test runs were done with non-mutagenized cells to develop the selection procedure i t s e l f . i s included i n Table IV.  One of the successful test runs (4b)  Out of 2x10^ c e l l s , 210 survivors were found  following 36 hours of light exposure.  Thus the frequency of wild type  survivors that was expected to survive the screening procedure (considered as experimental "noise") was about one c e l l per thousand. The f i r s t real test of the procedure came i n experiment 5a (Table IV) where approximately one million animals were run through the entire system yielding about 1000 survivors (447 were isolated and 18 saved as putative t s mutants).  Two weeks later, the remaining mutagenized cells,  which had been kept at 17°G i n stationary phase, were selected with no survival.  This was the f i r s t indication that the screening procedure was  capable of k i l l i n g a l l cells i n a run.  Since the system could be harsh,  mutagenized c e l l s were subsequently subjected to the selective system as soon as possible after treatment to reduce loss of mutants. The number of animals that survived the screening procedure varied extensively between runs (Table IV).  Selection 6a was an extreme example  of this inconsistency (almost 10^ c e l l s survived).  During the irradiation,  two classes of c e l l s were seen (swollen cells and normal cells) which must  17 have been caused by abnormalities i n the technique.  There were three  p o s s i b i l i t i e s to explain these abnormalities; poorly BU substituted bacteria, low amount of b a c t e r i a fed to the paramecia before i r r a d i a t i o n (this was not the case i n run 6a as strong t u r b i d i t y was noted i n the culture due to excess b a c t e r i a ) , or slow-growth of animals before the bacterial feeds food-vacuoles  In the l a s t case animals may  as usual.  have not formed a.s many  The classes (one badly damaged, the other  undamaged) may have been caused by a threshold of l i g h t damage to BU substituted DMA  i n the p o l y p l o i d macronucleus.  By t h i s point i n the  study i t was obvious that standardization of the parameters c o n t r o l l i n g the screening system had to be stringent. Standardized culturing of BU l a b e l l e d b a c t e r i a was (see Materials, and Methods).  c a r e f u l l y followed  Other parameters of the system, however were  adopted more or l e s s a r b i t r a r i l y , and were allowed v a r i a t i o n ; f o r example, the e q u i l i b r a t i o n time (the period that animals were heated to 3^»5°C before feeding of substituted b a c t e r i a ) , a,nd the duration of exposure to BU substituted b a c t e r i a .  An e q u i l i b r a t i o n time equal to one c e l l cycle ( s i x  to eight hours) was used because any block i n e i t h e r maintenance or i n i t i a t i o n of DNA  synthesis should have been encountered during that time  at the r e s t r i c t i v e temperature.  For example, an i n i t i a t i o n mutant i n S  when the temperature s h i f t occurred, should continue i n i t s c e l l cycle u n t i l the next G-^-S  boundary before h a l t i n g growth.  The period of i r r a d i a t i o n  was  reduced to about 12 hours following the experiments on the r e l a t i v e contributribution of vegetative and exautogamous l e t h a l i t y on the o v e r a l l selective l e t h a l i t y (total k i l l ) : of the Results.  the data w i l l be presented i n Section I I I  On occasion, the c e l l s were given time a f t e r i r r a d i a t i o n  i n a non-nutrient medium before i s o l a t i o n .  By leaving the animals i n  Dryl's s o l u t i o n overnight, many subviable i n d i v i d u a l s were eliminated.  This  18 reduced the number of single isolations to be made. Generally, abnormal looking c e l l s soon perished under these conditions while normal fast swimming cells did not. In total, more than 1300 lines were isolated, yielding 80 lines that passed the f i r s t series of ts tests.  From these, 29 were tested for  thymidine uptake using non-synchronous cultures; 10 showed decreased DNA incorporation below the 50 percent mark (four had 10% or less incorporation) at the restrictive temperature (Table Va).  From 13 lines tested, none  showed protein synthesis below the 50 percent level (Table Vb); none of the lines had as much as a 25 percent depression in the level of food-vacuole formation at 35°G (Table Vc, part l ) .  In another test on 11 lines (Table Vc,  part 2), l i n e number 132 showed no food-vacuole formation at the restrictive temperature.  This was the only observed case of such a deficiency. One  would expect that the food-vacuoleless category would be large because of the complexity of the process.  Not only must membrane and cortex be synthesized  to produce food-vacuoles, but control of the oral ciliature, and oral morphogenesis i t s e l f are involved (Orias and Pollock, 1973)* In summary, out of 1300 survivors of the selection procedure that were isolated, 0.3 percent appear to be conditional mutants of DNA metabolism. After a l l ts tests, there were 29 potential mutants, and of these, 14 percent were called ts DNA deficient.  TABLE Va, PART 1 PUTATIVE MUTANT GROUP ANALYSIS INCORPORATION OF 3H-THYMIDINE AT 27 AND 37°C  H N N N O o - o i n o • • • • o o o o o  O o • o  O H O O O o o o o o o • • • • • • o o o o o  c°wo o \ o 4 *o\o co n n n o H O H O H ^ ^ n o O O r i  • • • • • • • • •O•N•? \•  OJ r \ l T | 4 O H o  o o o o o o o o o o o o  H^COV>OOOONONO OOOU^OOOONOOU^O • • • • • • • • • • • • o o o o o o o o o o o o  N H N V\0 C^OO CO O CM C^CO  • •••••••••••  C O  H H C V l H N N H C O H O O p + l + l +1 +1 +i -H +1 +1 + i + i -Fl - f l O H ONCNoiJ-vO C\! ON cn CO CO  o  • ••••••••••• (3  COOHO\(MCONON\ONNO\ • • • • • • • • • • • • (^N O N 0 0\^ O O O O O +1 +1 -H -H +1 H  H  +«4^i^  rH  -5F - 5  +1+1+1+1 rH  N [ S H ( ^ > ^ C O N O H r>0 C (NiCVJvovOvOvOO-COCOONO O rH O  S  •H -P  COp  -p o  o  •H  -P  C D M+> c°\ TH 6 c o era • > J3o c c o o O C D >>,H > co x: co -I— -P o C D o rH ci 0) >> (1) c •rH X) 3 C D . o T H o - P co to  t  •2 o o  5  •H -  - P  See  a o  O H-> rH * x: o co  CO  s  C O rH  a  +>  Line  Cont 1  66 85 90 101 123 124* 125 1* 24 32 42 55 102 103 126 127 128 132  Cont 2  Median Rank # at 27°G  5 5 4-5 4 „ 0-1 1-2 4 4 3-4 4 3 3-4 4 0-1 4 3-4 1 2 1 4  Median Rank # at 35°G  4 1 2-3 2 1-2 5 0 4 0-1 3 2-3 3 3 0 3 2 1 0-1 0-1 3  Description of Ratio 35/27°G low low low low high high v. low equal v. low low low-equal equal-low low equal-low low low equal low low low  Table Va, Part 2. ^H-Thymine incorporation was scored on a ranking scale to identify low incorporation at 35°C. The median category i s shown at both temperatures. The ratio of 35 to 27°C incorporation was assigned a description! very low (V. low), low, equal, or high. Very low occurred when the line incorporated l i t t l e or no DNA precursor at 35°G« 124* & 1*, lines 124 and 1 appeared to have the largest decrease i n incorporation at the restrictive temperature, therefore grain counts were made (part 3)»  TABLE Va, PART 3 PUTATIVE MUTANT GROUP ANALYSIS INCORPORATION OF 3H-THYMINE AT 27 AND  I I  O -P  ocvj ro  «* 8 W -P O. CM  r H VO O VO • • H H O •  CD  S  O O UN CO  one  •H  O O  O - rH • • O  O  rt  +> m 0)  £ 3 3 O  *  •  O O O  CV!  O O O O O O • • • O O O  P?  • CD  CO  f J+l £ 0 10  & 0  W WN O CN C O ^  CD  cc  CQ  •H rt 4.1 S (D  T I  go & I O co*  evi  CD  O  a  UN UN O v  o  • •• CVJ +1 r H +\ O +1 CO UNVO  • « •  H  CM C O O N  • >t •  -ff CO CM t! t i +' Ov O v O  •  • •>  CV-3- -3"  CO CO CVJ  -P C CVJ O rH O  CD  35°C  Line  22 27 51 63 65 66 68 77 80 85 90 101 125 Cont 1 Cont 2  -'H-leucine 2?°G Mean Incorp. 4 SE. 21.4±1.5 18.6±1.1 18.4±1.1 16.4±1.3 18.3±1.4 16.3±1.0 17.340.9 17.8±0.9 11.3±1.0 17.9±1.6 14.84L.0 13.5*0.6 16.84L.0 32.0+2.4 19.441.4  >  ^H-leuclne 3?°G Mean Incorp. 4 SE.  V  Ratio of 27 & 37°G Means  Ratio of 27ts & 27wt Means  18.440.9 18.1*L.l 17.8±1.0 18.6*L.2 17.2tL.2 9.2±1.2 23.5±1.1 17.8±1.2 15.5±1.2 9.5±1.1 13.6±1.4 l?.8±l.i 13.34L.0 19.141.2 13.2±0.8  0.09 0.79 0.71 0.18 O.63 0.00 0.00 0.71 0.92 0.01 0.38 0.00 0.05 0.00 0.00  0.86 0.97 0.97 1.13 0.94 0.56 1.36 1.00 1.37 0.53 0.92 1.32 0.79 0.60 0.68  1.08 0.93 0.92 0.82 0.91 0.82 0.86 0.86 0.57 0.84 0.74 0.67 0.84 1.00 1.00  1  Table Vb. Incorporation of protein precursor at 27 and 37°G. descriptions from Table Va, part 1 apply i n t h i s table.  All  Line  Food-vac. 23°C Mean Form'n + SE.  Ratio of 23 & 35°G Means  Food-vac. 350C Mean Form'n t SE.  51 65 66 68 80 85 90 100 101 123 124 pwA Cont  7.7±0.7 5.5±0.7 9.0+0.9 9.3^0.7 8.6±0.6 7.ltL.2 6.0±0.9 9.4±0.8 9.9^0.8 10.3±0.8 8.9±0.7 7.1*0.4 7.9*1.0  Line  Approx. Ratio of 23 to 35°G Means  9.2+0.6 5.8±0.6 6.8+0.6 9.6+0.7 9.3*0.8 5.2±0.4 6.4i0.3 8.li0.5 8.3*0.5 8.4±0.5 8.1*0.6 8.1*0.6 6.3*0.8  0.60 0.09 0.69 0.42 0.01 0.05 0.52 O.38 0.10 0.19 0.88 0.93 0.23  1.27 1.13 0.80 1.09 1.15 0.78 1.13 0.92 0.89 0.87 0.97 1.21 0.80  Ratio of 27ts & 27wt Means 0.76 1.06 0.86 0.93 0.97 1.15 0.88 0.98 0.91 0.89 1.07 0.9^ 1.00  0.06 0.48 0.24 0.71 0.97 0.48 0.98 O.76 0.59 0.43 0.55 Oi-88  **) o o  V >  a o  *j S  I  t-3  s  63 125 1 24 32 103 126 127 128 132  1.50 1.00 0.50 1.00 1.00 0.50 0.50 0.66 1.00 0.00  o ro  Table Vc. Food-vacuole formation a t 23 and 35°G. Both means are given along with standard errors, probability that the F s t a t i s t i c «= 1, and the r a t i o of formation at high and low temperatures. These data were standardized against wild types which gave a r a t i o of Wo (35/23°G means). The second part of Table Vc shows approximate food-vacuole formations f o r some putative t s l i n e s .  O O  N> Vol  TABLE Vd PUTATIVE MUTANT GROUP ANALYSIS GROWTH AT 27°G (FRACTION OF CONTROLS)  Line  G  51 63 65 66 68 80 85  0.97 1.04 1.04 1.04 1.11  1.01 1.11  0.81  24  32  Cont  t  0.91  125 1  128  a  0o94 1.08  124  132  27° C  1.00 0.75  100 101 123  55 102 103 126 127  °Son  1.08  90  42  r  '  0.91 0.88 0.92 0.91  0.54  0.71 0.94 1.08 0.91  1.08  1,00  Table Vd,. V i a b i l i t y index, growth a t 27 C standardized against c o n t r o l s (set t o 1.00). The index f o r the l i n e s was c a l c u l a t e d as a mean o f f i s s i o n r a t e s over 10 days; the w i l d type c o n t r o l s averaged 4.43 f i s s i o n s per day.  25 II  E f f i c i e n c y o f the S e l e c t i o n System (Marked-Recapture Experiment) The screening system was c a l i b r a t e d by marking a known t s mutant  ( l i n e #2A2, i s o l a t e d through the screen) with the behavioural gene pawn.^ Selections were c a r r i e d out s t a r t i n g with* 1, 10, o r 100 pawn t s double 4 5 , 5 mutants i n 10 , 10 , andtox 10^ w i l d type c e l l s .  Recovery r a t i o s ranged  2 4 3 from 3»4 x 10 t o 1.2 x 10 , the mean over seven runs being 7»6 x 10 J  (Table V l ) .  Thus, under average c o n d i t i o n s with l i t t l e d e s s i c a t i o n ,  absence o f crowding, and good q u a l i t y l a b e l l e d b a c t e r i a f o r food, the screen enriched f o r t s mutants o f the k i n d d e s i r e d , by about 7500 times. TABLE VI EFFICIENCY OF THE SELECTION SYSTEMt (MARKED-RECAPTURE EXPERIMENT) Run //  Entrance Ratio  E x i t Ratio  Exit/Entrance Ratio  1  100pw/l.2xl0^/T pw/WT.=8.34xl0"3  25pw/NIL WT pw/WT.*25.0  >3.0xl03  2  100pw/l.2xl0**WT pw/WT.=8.34x10-3  I6pw/NIL WT pw/WT.*16.0  > 1.9x103  . Wo  100pw/l.2xl05wT  pw/WT.=8.34x10"^  45pw/l6 WT pw/WT.=2.82  , 3«4xl0  Wo  4.  100pw/6.0xl05wT pw/WT.=1.67x10"^  8pw/7 WT pw/WT.=l.l4  6.8x103  8%  5  10pw/l.2xl0 % pw/wr.-s^xio-^  pw/WT.*6.0  6pw/NIL WT  > 7.2x103  60%  6  lpvr/l.2xloVr pw/WT.=8.34xl0"5  2pw/3 WT pw/WT.=0,67  8.0x103  200%  7  ipw/i.2xioVr pw/WT.=8.34xl0~5  IPW/NIL vrr  >1.2x10^  100%  3  .  Z  2  25%  pw/WT."1.0  Table VI. Range over 7 runs was 3 . 3 8 x l 0 to 1.2xlO K 7.6lxl03 pw=pawn marked ts2A2 c e l l s , WT=wild type c e l l s . 2  o  Recovi  Pawn •  L  The mean being  26  III  Pattern of Decrease i n Survival and Clone Forming; Ability Due to Selection Just as temperature-sensitive periods usually precede death i n most  developmental lethal ts mutations, for example, i n Drosophila melanogaster (Suzuki, 1970), so i t was expected that a dose of UV light large enough to destroy the c e l l s ' a b i l i t y to form clones would be passed long before the cells actually diedo  To test this, c e l l s were subjected to varying  exposures of l i g h t i n otherwise standard selection runs.  Cell density i n  the cultures was determined, and individual isolations were made. The vegetative lethality and clone forming a b i l i t y were plotted as functions of exposure time (Figure 4 ) . The number of cells dropped below the 50 percent mark after 13 hours irradiation, but the clone forming a b i l i t y had reached the level at four hours.  After approximately 12 hours the  ability to form a clone was n i l , although individual cells survived beyond 24 hours.  It was apparent that cells were f a r more heavily damaged than  was obvious during the selection treatments.  On the basis of these  observations, l i g h t treatment was restricted to 12 hours.  27  FIGURE 4.  Vegetative survival and clone formation with varying UV light exposure. FIGURE  200-  4 A (vegetative survival of BU substituted cells)  HOURS LIGHT EXPOSURE  28 IV  Relative Contribution of Components of the Selection System to Total Cell Death Because two c r i t i c a l periods i n the selection procedure had been  discovered (the threshold of cloning a b i l i t y failure and death of i n i t i a l cells) there was reason to assess the contributions of various components Of the selection system to the composite pattern of c e l l fate.  Firstly,  experiment 6a (Table IV) was studied i n detail because i t resulted i n comparatively l i t t l e vegetative death but made i t possible to examine the effects of BU alone and BU plus UV l i g h t on the amount of exautogamous death and i t s role i n the selection procedure.  Secondly, the effect of UV  light alone on vegetative survival was examined.  It was reasoned that i f  few cells died during or soon after the irradiation, many might be lost following the subsequent autogamy.Further, two definite classes of c e l l shapes other than normal were noted during the light exposure:  the f i r s t  was the "rounded-up" c e l l appearance that was known to precede l y s i s ; the second was slightly pear shaped and vacuolated. expected to die much later.  The pear shaped cells were  Three hundred and twenty-three isolates were  made and cultured through autogamy. The results (Table Vila) showed that vegetative death increased from wild type control (2.9/S) through "dark" control  (5*6%)  to experimentals  (17%)•  Following autogamy, at least a two  fold increase i n death was realized i n a l l groups. exautogamous survivors was then checked.  The v i a b i l i t y of the  Wild type survivors grew well (all  viables); however i n the "dark" controls, seven percent of the survivors f e l l into the subvlable group. 17.2  The experimentals were even worse than expectedi  percent of them were subviable.  If the number of subviable lines i s  summed with the exautogamous deaths, i t i s possible to see the total effect of the components of the screen. The wild type exautogamous death and subviability (11.8$) was increased to 35»0 percent by BU, and to 58.6 percent when irradiated.  The influence of BU on micronuclei as distinguished from  29 that of UV light can also be seen at a l l points i n the selection (Table Vllb). Both influences (BU, UV light) decrease the v i a b i l i t y of exautogamous survivors by 23.2 and 23*6 percent respectively; these cells were either k i l l e d or rendered subviable.  I t appeared that the effect of both BU and  of light was to preferentially k i l l rather than disable.  The possibility  that light alone might have a detrimental result on the cells was tested; for no "light-alone" controls were used i n the previous experiment. TABLE VII RELATIVE CONTRIBUTION OF COMPONENTS OF THE SELECTION SYSTEM TO TOTAL CELL DEATH Exautogamous Viability Group  Vegetative Death  Wild type (no treatment) Dark cont. (BU, no UV light) Experimental (BU, UV light)  Exautog, Death  Survivor Viability* . SubViable viable  Exautog. Death+ Subviable  2.9$  11.8$  0  88.2$  11.8$  5 M  28.0$  7.0$  65.0$  35.0$  41.4$  17.2$  41.4$  58.6$  2,7%  16.2$  7.0$  -23.2$  23.2$  11.4$  13.4$  10.2$  -23.6$  23.6$  17,0%  B Actual DarkBU Effect (Dark minus wild type) Actual UV Light Effect (Exptl. minus Dark)  Table VII-A, breakdown of c e l l fate i n the Selection System. *, clones were scored on a ranking scale - see Materials and Methods. Exautogamous death, and.Survivor v i a b i l i t y categories add up to 100$. Table VII-B, actual isolated effects of bromouracil and of UV light (after BU treatment) calculated by subtracting pairs of treatments (Dark control minus Wild-type control, and Experimental minus Dark control) from Table VII-A.  30 The influence of light alone was determined by irradiating wild type cells i n petri dishes with Dryl's solution; the cells were given 13 hours of light at two intensities (less than 3 cm. distance and 20 cm. distance).  One hundred and twenty-six isolations were made and grown to  test clone forming a b i l i t y .  Samples of the 13 hour exposed cells (both  intensities) were delayed i n Dryl's solution up to 45 hours to see i f dark repair of "light exposure damage" was possible since BU substituted mammalian cells show rapid loss of single stranded breaks i n the DNA (on alkaline sucrose gradients) following irradiation (Humphrey et a l . , 1970; Smets and Cornelius, 1971) as does Escherichia c o l i DNA (Ley and Setlow, 1972).  Another sample of cells was given 57 hours of high intensity light  at close range and then isolated; almost 60 percent cloning a b i l i t y was retained.  In the experiment, no significant loss occurred i n the samples  exposed for 13 hours (Figure 5)«  I t was concluded that the UV light emitted  by the fluorescent tubes was relatively harmless to the paramecia.  31 FIGURE 5 EFFECT OF LIGHT EXPOSURE AND DELAY BEFORE ISOLATION ON CLONE SURVIVAL  CD  § 0'  10 20 30 40 HOURS OF DELAY BEFORE ISOLATION  50  Figure 5. The percent of clones surviving 13 hours of UV light exposure with up to 45 hours delay before isolation (no BU substituted bacteria were fed). O , control (no light); X, high intensity light; low intensity light.  32  DISCUSSION Mutagenesis and the Selection System. The aim of the mutagenesis procedure was to achieve 40-60 percent post-autogamous lethality.  Presumably, this should provide reasonable  levels of recessive mutations without a high percentage of multiple hits. However i n the light of work by Guerola, Ingraham, and Cerda-Olmedo (1971), this may be an oversimplification of the problem.  In a study of NG  mutagenesis i n Escherichia c o l i . they found that as many as 50 base changes occurred within two minutes map distance about a specific selected mutation. Thus, because NG causes clustering of mutagenic damage, induced phenotypes could be due to a composite pattern of multiple h i t damage rather than a single point mutation. If this i s the case, slow-growing putative mutants should not necessarily be discarded unless they are incapable of mating.  A few  successive matings should remove most unlinked, or loosely linked subviable lesions.  This type of clean-up i s well described i n Sonnebom's methods  paper (1970) but because i t i s time consuming the procedure was not followed in the present study. Several mutagenesis experiments were conducted with good results: there was l i t t l e loss of animals through toxicity of the mutagen or through harshness of handling (see Section on mutagenesis i n Materials and Methods). Indeed, mass cultures often may have doubled i n number shortly after the treatment (e.g. mutagenesis #10, Table IV) implying that the cells were growing, and that a portion of them should have been i n the S phase of the c e l l cycle.  There i s no macronuclear G^ i n Paramecium aurelia syngen 4  (Berger, 1971) and therefore a large number of cells must have been arrested in S and released after the 60 minute mutagenesis.  The mutagenic effect of  NG i s highest i n the S period of Escherichia coli (Cerda-Olmedo, Hanawalt,  33 and Guerola, 1968), i n fact i t halts DNA synthesis immediately in that c e l l (Guerola, Ingraham, and Gerda-Olmedo, 1971).  Cells w i l l presumably be  affected by NG no matter what their stage i n the c e l l cycle; as well as associating with the replication region, the mutagen has a marked detrimental effect on protein synthesis, and a lesser inhibitory effect on RNA synthesis in Escherichia c o l i (Cerda-Olmedo and Hanawalt, 1967).  Studies on the growth-  inhibitory characteristics of NG on mouse tumour cells (Leiter and Schneidermann, 1959;  Goldin et a l . , 1959;  Skinner et a l . . I960; and Greene  and Greenberg, i960) indicated that effects of the chemical on eukaryotes might be similar to those on bacteria.  These studies were published before  the mutagenic effect of NG was known. Thus the increased effectiveness of the mutagen during DNA synthesis may be partially or wholly offset by i t s inhibitory effect.  However, since only about 18 percent of the population  would be expected to be i n micronuclear S period during exposure to the 11 mutagen,  this effect would be relatively small.  Mutation i n macronuclear  DNA was perhaps more common than i n micronuclear since the S period i s five times as long (occupying approximately 0.70 of the c e l l cycle).  This implies  12 that almost 61 percent of the cells were treated i n macronuclear S.  However  since there are over 800 copies of the genome being replicated and since the duration of treatment was 60 minutes, the chance of a l l copies of an essential gene being functionally altered seems small.  If a sufficient number  of copies of an essential gene were damaged, then the particular c e l l line would i n a l l probability have become a "slow grower" u n t i l the subsequent autogamy when the cells would probably have died due to micronuclear damage. After vegetative death few cells were lost u n t i l the completion of phenotypic lag. after autogamy. Sonneborn (1965) discussed the concept of "silent mutations"; those mutations which change DNA codons i n such a way that the new triplets s t i l l  34 code for the same amino acid.  He reasoned that up to 20 percent of a l l  single base changes might f a l l into this category.  Whitfield, Martin,  and Ames (1966) discovered that only one percent of mutations induced i n Salmonella typhinurium the HisC gene of actually resulted i n a new phenotype (auxotrophic for histidine).  This extreme silence of mutations may not  hold for the entire bacterial genome, but the overall figure may be high. If these estimates are accurate, many of the multiple hits (typical of NG treatment) may be harmless to the micronuclear information of the organism.  The prospect of "silent mutations" coupled with the presence  of 800 macronuclear copies of the genome (ready to give an intra-macronuclear complementation effect) make Paramecium a resistant c e l l to mutagenic damage. There was probably a large number of copies of each mutation i n the cultures that were selected because homozygosed cells had to be grown for eight to 10 c e l l cycles after autogamy before expression of mutant phenotypes occurred (phenotypic lag). beneficiali  This duplication of mutants may have been  since the incidence of recovery of a mutant (as determined by  the Marked-Recapture Experiments - Section II of the Results) varies, and can be as low as eight percent, the presence of a significant number of siblings i n the culture was not altogether undesirable. The recovery of a particular mutant by the screening procedure required the cells to withstand a minimum of 12 hours of the restrictive temperature; thus rapidly lethal mutants were lost. Once the treatment with NG was terminated, the populations were immediately divided into batches - usually four to five 5 ° ml. cultures. These batches were grown to phenotypic expression separately. The chance of siblings occurring between batches was therefore small, however within batches i t was expected to be high.  The division of post-treatment cultures  hopefully would yield complementation groups useful i n initiation of routine  35 crosses to be performed later. not within batches.  The f i r s t crosses should use lines between,  The number of sibs present i n a batch after a certain  time depended on the length of the period of growth between homozygosis and selection, and the rate at which that mutant divides. allowed 10 fissions, i t follows that there was up to 2^  I f this period copies (sibs) of  a single mutational event present i n the culture. In reality, this i s unlikely because wild type cells w i l l grow faster than many mutants at 27°C. Contamination by wild type cells or cells of the opposite mating type during the selection runs would have lowered the efficiency of the 13  screening procedure.  Induction of a selfing line  J  by mutagenesis could  have also caused mating (and consequent heterozygosis of recessive lesions). For this reason, i t was advisable to use mating type VIII paramecia (as was done i n a l l cases), because selfing clones are only known to arise from mating type VII (Butzel, 1953)*  Contamination disasters of a l l origins  would presumably not result i n an increase i n survivors, but a decrease i n the number of putative t s lines recovered. While the selection system presumably worked through the induction of damage to the BU substituted DNA, the possibility that photolysis of BU substituted mitochondrial DNA had an influence, could not be excluded. Obviously, several parts of the mutagenesis and screen set-up might have affected these v i t a l organelles. The effect of NG on mitochondrial DNA i s unknown, as i s the extent of damage due to UV light photolysis of BU substituted organellar DNA.  However, chloramphenicol and erythromycin  resistant mutants controlled by cytoplasmic determinants (mitochondrial) have been isolated following NG treatment i n Paramecium aurelia (Beale, .1969; Adoutte and Beisson, 1970, 1971; and Beale, Knowles, and Tait, 1972); and i n Tetrahymena piriformis. (Roberts and Orias, 1973).  As other effects  36 of NG, one might expect disruption or loss of function i n the organelles. This, however, could not be the case, because mutant l i n e s survive the treatment.  I t i s possible that most mitochondria were regularly destroyed  but sufficient numbers remained functional to allow the c e l l s to metabolize at a reduced rate. The p o s s i b i l i t y that most of the mitochondria were damaged i s not supported by observation; most c e l l s that survived grew well immediately a f t e r l i g h t treatment. Thus t h i s factor could not have been overwhelming. One of the advantages of the experimental design was that survivors of an i n e f f i c i e n t selection (due to poorly l a b e l l e d b a c t e r i a l food perhaps) could be grown s l i g h t l y to clear the c e l l ' s metabolic pools of BU, and then retreated with BU substituted bacteria and irradiated again, to enrich f o r putative t s mutants.  (Generally, a successful run l e f t so few survivors  that reselection was t o t a l l y unnecessary.) Whether reselection made the overall system more e f f i c i e n t was not c r i t i c a l l y tested.  However i n  selections 10a to lOe (Table IV of the Results), the percentage of ts l i n e s 14 i n isolates was s i g n i f i c a n t l y higher i n the reselected runs. The p o s s i b i l i t y of reselection raised the question of simplification of the procedure to reduce work. Because 30 percent of exogenously fed H-thymidine i s incorporated into the macronucleus (Berger and Kimball,  1964;  Berger, 1971), i t may have been possible to use the less specific exogenous BU feeding of the batch cultures. This type of feeding, without b a c t e r i a l vector, would probably have resulted i n various degrees of c e l l damage and death. Perhaps two classes of c e l l s would have been generated i n the selection as i n run 6a (Section I of the Results). Simplification of the Selection System might produce a larger group of survivors that f a l l into the category of wild type (often called "noise"). Such ts l i n e s , that incorporate more than 10 to 20 percent of the normal  37  amount of label into DNA at the restrictive temperature, make up the bulk of the screen survival. are d i f f i c u l t .  Explanations for the existence of the category  In some cases autoradiographic data demonstrated an "on-off"  characterT a sample of c e l l s from a particular line contained individuals K i t h either very low incorporation ("off"), or a higher level of incorporation ("on"). The inference i s that cells of such a line may be exhibiting less penetrance, or that the particular mutation confers a c r i t i c a l ts period on the c e l l s (that were unsynchronized), f o r example, i n i t i a t i o n .  If the c e l l  i s already i n DNA synthesis, i t should halt at the G^-S boundary.  Because  incorporation figures were based on sample means, an intermediate figure (between "on" and "off") was recorded. Rationale for the Selection System and Possible Applications 1.  The study of the mutants themselves and the analysis of their roles  i n the physiology of DNA synthesis i n Paramecium w i l l prove interesting.  The  Selection System only demands that the ts c e l l stop synthesizing DNA within one c e l l cyclei however, mutations that act much faster may be present. Other studies concerning related functions of the c e l l could be undertaken; for example, cytokinesis, and food-vacuole formation may be defective as well as DNA synthesis. 2.  '  '  _  It should be possible to do experiments on the regulation of total  DNA content i n Paramecium. For example, a shift to the non-permissive temperature before termination of macronuclear DNA synthesis in a ts DNA maintenance line could be used to examine the precision of regulation of DNA content.  It might be possible by early terminations or replication over  several c e l l generations to produce a c e l l with perhaps 70 percent of the normal DNA content before the c e l l reacts to the problem. be easyj  This would not  i t would be necessary to shift a series of synchronized cells to  38 the r e s t r i c t i v e temperature just before termination of DNA synthesis.  At  each successive c e l l generation a set of synchronized c e l l s would have to go through the same treatment. Representatives from each generation would be Feulgen stained and the r e l a t i v e amount of macronuclear DNA measured by microdensitometry (macronuclear DNA content would be estimated r e l a t i v e to the amount of micronuclear DNA observed). In Tetrahymena piriformis , Cleffmann (1968) demonstrated that a decrease i n DNA content occurs normally u n t i l about 70 percent of the normal i s reached, then the c e l l performs an additional doubling (up to the lk0% level) and starts a downward trend again, a l i t t l e with each c e l l cycle.  The steplike regulation may be present i n  Paramecium, but -the threshold may be higher. 3.  Ts DNA d e f i c i e n t mutants could be useful i n studying the i n i t i a t i o n of  DNA synthesis i n macronuclear anlagen and macronuclear fragments i n exconjugants.  In addition, the mechanism by which fragment DNA synthesis i s  normally selectively inhibited (Berger, 1973) autogamy could be examined.  A t s DNA  following conjugation or  mutant would allow manipulation of c e l l  nuclei resulting i n a cessation of DNA synthesis i n the macronucleus of a c e l l that had macronuclear fragments of the genotype ts/+, and micronuclei, t s / t s . A temperature s h i f t up to the r e s t r i c t i v e l e v e l a f t e r conjugation may force the fragments of the o l d macronucleus (a r e l i c of the preconjugation macronucleus) to synthesize DNA, and form a substitute nucleus. The activation of the o l d fragments Would depend on a fast i n h i b i t i o n of the macronucleus by a ts mutation with no other deleterious effects.  The experiment would therefore  mimick the rare natural phenomenon known as "macronuclear regeneration".  The  design of the proposed procedure i s more f u l l y described i n Appendix I I . 4.  Ts mutants might allow synchrony of mass cultures f o r c e l l cycle  studies.  The usefulness of synchronized cultures has been well established.  Hotchkiss (1954) developed a method f o r synchronizing the growth of bacteria,  39 and Scherbaum and Zeuthen (1954), using heat shock achieved 85 percent synchrony i n Tetrahymena.  These procedures opened new avenues to the  study of DNA metabolism i n Escherichia c o l i (Schbach and Davern, 1970; Rodriguez, Dalbey and Davern, 1973)  and to the study of metabolic control  and oral morphogenesis i n Tetrahymena (Frankel, 1967)»  Most recently,  Hartwell and Hereford (1973) used ts mutations to synchronize yeast cells although somewhat unsuccessfully. Paramecium.  No such breakthroughs have occurred i n  Obviously, instead of labourious manual collection of dividing  cells, synchrony could be realized by use of a DNA deficient mutant that would suspend growth at a particular point i n the c e l l cycle.  Exautogamous  or exponential cultures could be heat shocked for six to 12 hours (the time spent at the restrictive temperature i n the Selection System).  Once returned  to the permissive temperature, the degree of synchrony of the f i r s t division may be substantial.  Any amount of synchrony would be a help i n the collection  of dividers. Concluding Statement In summary, c e l l lines exhibiting a l l levels of depression i n DNA synthesis have been isolated u t i l i z i n g the selection system defined.  Four  ts lines with less than 10 percent incorporation at 35° C (representing 20$ of the putative lines tested) have been isolated; possible applications of these ts lines are diverse.  Other attempts i n eukaryotic organisms have  yielded poorer results using replica-platingt  i n Ustilago. Unrau and  Holliday (1970) tested 400 ts mutants and found only five (l$) affecting DNA synthesis - none of these was completely blocked.  In yeast, Hartwell  (1967) obtained only four ts DNA deficient mutants out of 400 tested, again 1 percent.  Therefore, the yield from the Selective System (l4$) presented  here for'Paramecium appears to be relatively efficient and can be improved with more comprehensive t s tests prior to incorporation studies.  40  NOTES  1.  "Ts m u t a n t " i s u s e d even though no g e n e t i c a n a l y s i s was p e r f o r m e d because a l l t e s t e d c e l l l i n e s have been s u b s e q u e n t l y c o n f i r m e d a s gene c o n t r o l l e d ( r e c e s s i v e ) m u t a n t s .  2.  O v e r n i g h t 10ml. c u l t u r e s were supplemented w i t h JO/jg/ml* of thymine. O n l y lQtfg/ml. was u s e d i n t h e b u l k 750ml. c u l t u r e s making e l i m i n a t i o n (by c e n t r i f u g a t i o n w a s h i n g ) o f t h e p y r i m i d i n e more complete b e f o r e t r a n s f e r of the b a c t e r i a t o 5 - b r o m o u r a c i l .  3.  BU s u b s t i t u t e d b a c t e r i a were f o u n d t o be p o t e n t f o r use i n t h e s c r e e n i n g p r o c e d u r e even a f t e r f i v e days a t 4°C p r o v i d e d they were s t o r e d i n t h e i r own growth medium; t r a n s f e r t o D r y l ' s s a l t s o l u t i o n p r i o r to r e f r i g e r a t i o n destroyed the c u l t u r e .  4.  Thymine s t a r v e d c u l t u r e s w i l l r e c o v e r i f r e s t o r e d t o medium. (Donachie and H o b b s , 1967).  5.  The d e f i n i t i v e p r o o f t h a t BU e n t e r s t h e macronucleus (and m i c r o n u c l e u s ) m i g h t be a t t a i n e d by u s i n g ; A , r a d i o a c t i v e l a b e l l e d BU (Bromine i s o t o p e ) w h i c h i f d e b r o m i n a t e d , would n o t l a b e l DNA. The BU must b e i n f l u e n c e d by b a c t e r i a l p o l y m e r a s e and n u c l e a s e s and p o l y m e r a s e s i n t h e p a r a m e c i a - i t i s p o s s i b l e t h a t t h e BU i s d e b r o m i n a t e d i n b a c t e r i a o r p a r a m e c i a ; d e o x y u r i d i n e would go i n t o DNA and y e t be u s e l e s s f o r UV d e a c t i v a t i o n ( d e o x y u r i d i n e i s a v e r y e f f i c i e n t DNA p r e c u r s o r b e c a u s e i t can be c o n v e r t e d t o t h y m i d y l i c a c i d by thymidylate s y n t h e t a s e ) . B , t h a t BU g e t s i n t o t h e c i l i a t e DNA by t h e d e s c r i b e d t e c h n i q u e s , m i g h t be shown by s p i n n i n g down DNA f r o m t h e a n i m a l s on CsCl2 d e n s i t y g r a d i e n t s t o d e m o n s t r a t e t h a t t h e heavy b r o m i n e s u b s t i t u t e d s p e c i e s i s p r e s e n t .  6.  A p r o t e c t i v e e n c l o s e d p l e x i g l a s s g l o v e - b o x was b u i l t t o ensure s a f e h a n d l i n g o f t h e mutagen. T h i s box was u s e d w i t h i n a fumehood.  7.  G e n e r a l l y , t h e t r e a t m e n t s o l u t i o n s were t o p p e d - u p c a r e f u l l y w i t h d i s t i l l e d water. Change i n o s m o t i c p r e s s u r e was r i g o u r o u s i f t h e l e v e l was r e p l e n i s h e d t o o q u i c k l y . One a t t e m p t was made t o m a i n t a i n t h e f l u i d l e v e l i n t h e p e t r i d i s h e s a t i t s s h a l l o w e s t (which r e d u c e s s o l u t i o n a b s o r p t i o n o f t h e l i g h t b e f o r e t h e c e l l s were i r r a d i a t e d ) w i t h o u t r i s k i n g t h e d e s i c c a t i o n when l e f t u n a t t e n d e d f o r o v e r s i x h o u r s . ' The a n i m a l s were exposed a s u s u a l , b u t a c o n s t a n t f l u i d d e p t h a p p a r a t u s was d e v i s e d b a s e d on t h e s i p h o n , however i t was n o t w i d e l y implemented b e c a u s e o f u n r e l i a b i l i t y .  8.  I f t h e t h r e s h o l d was n o t p a s s e d d u r i n g o r s h o r t l y a f t e r l i g h t t r e a t m e n t , t h e c e l l s t i l l had a n enormous o b s t a c l e a h e a d : autogamy w i l l be t h e Nemesis o f many i n d i v i d u a l s . ( T h i s argument o f c o u r s e h o l d s f o r m i t o c h o n d r i a , e x c e p t t h a t t h e t h r e s h o l d f o r each o r g a n e l l e must be e x t r e m e l y low - b u t t h e number o f o r g a n e l l e s i s h i g h . )  sufficient  9. A c r o s s o f pawn/pawn x ts2A2/ts2A2 was made by E r i c P e t e r s o n .  E x c o n j u g a t e s o f t h i s c r o s s were u s e d t o p i c k up F£ d i h y b r i d p r o g e n y . Ts 2A2, i s o l a t e d by t h e s e l e c t i o n s y s t e m , i n c o r p o r a t e s l i t t l e o r no  41 DNA precursor a t 35°C. T h i s t s a l s o stops growth a f t e r about f i v e hours a t the r e s t r i c t i v e temperature ( E r i c Peterson, personal communication). 10.  Since the v e g e t a t i v e macronucleus i s h i g h l y p o l p l o i d , the c e l l could cope with s e r i o u s g e n e t i c damage, having other untouched copies of genes, u n t i l autogamy when the new macronucleus i s made from the d i p l o i d germinal micronucleus.  11.  In an e x p o n e n t i a l l y growing asynchronous c u l t u r e , the p r o p o r t i o n of c e l l s t h a t are i n micronuclear DNA s y n t h e s i s can be approximated by use of the equation tm/T = M/0.693 o r M X 1.44 (these equations have been used by Hoffman, 1949; Stanners and T i l l , I960; Smith and Dendy, 1962; and P a i n t e r and Marr, 1968| and are based on the curve y = 20-~ ) where M = m i t o t i c index, t = c e l l c y c l e time, and tm «* the time i n m i t o s i s a t the end of the c e l l c y c l e (tm can be used i f M i s s m a l l ) . Therefore, M = 35/300 minutes = 12 percent, tm/T = 0.12/0.693 = 0.12 x 1.44 = 0.173. According to these c a l c u l a t i o n s presumably 17 percent of the c e l l s would be i n micronuclear S a t any moment. T h i s i s o n l y an approximation s i n c e S f o r Paramecium m i c r o n u c l e i i s not a t the end df the c e l l c y c l e (there i s a subsequent 115 minute G^). A more accurate estimate can be c a l c u l a t e d u s i n g the p l o t of y = 23-~ ) which g i v e s r e l a t i v e numbers of c e l l s a t p o i n t s i n the c e l l c y c l e (by c a l c u l a t i n g the area t h a t micronuclear S occupies under the curve). T h i s g i v e s a number of 18 percent f o r micronuclear DNA s y n t h e s i s . I f many c e l l s entered S i n the presence o f the mutagen, the f i g u r e representing the p o r t i o n of c e l l s t r e a t e d i n micronuclear S i s conservative. x  x  12.  Using the approximation M i t o t i c index x 1.44 f o r the c a l c u l a t i o n of the number of c e l l s i n macronuclear DNA synthesis g i v e s 0.70 x 1.44 = 1.01. However c a l c u l a t i o n of the area under the curve (y = 2 ( l ~ ) ) g i v e s 61 percent - a more r e a l i s t i c f i g u r e . T h i s discrepancy proves the inadequacy of the f i r s t estimate when the duration of S i s too g r e a t . x  13.  Byrne (1973) has i s o l a t e d mutations which, when homozygous, produce s e l f i n g . B u t z e l (1953) f i r s t proposed t h a t mating type substances (causing the mating r e a c t i o n ) were synthesized i n a pathway. Substance 0 (odd M.T.) was a precursor f o r substance E (even M.T.), where 0 was r e s p o n s i b l e f o r making an animal mating type V I I ; and E, mating type V I I I . Taub (1966) agreed w i t h these f i n d i n g s . Byrne suggested t h a t the a l l e l e s he had f r e s h l y i s o l a t e d (mtA, mtB, and mtC) could be b l o c k s along the pathway t o E substance. His f i n d i n g s are c o n s i s t a n t with the observation t h a t change of mating type i n c e l l s occurs i n the d i r e c t i o n 0 to E o r mating type VII to mating type V I I I . As an a s i d e , a system of d e l i b e r a t e mating, f o l l o w i n g mutagenesis i n s t e a d of autogamy, could be attempted to screen f o r dominants.  14.  AX t e s t o f a s s o c i a t i o n was conducted on the data from a l l s e l e c t i o n runs f o l l o w i n g mutagenesis 10 (Table 4 o f the R e s u l t s ) , to see i f there was a d i f f e r e n c e i n the number o f l i n e s i s o l a t e d i n s i n g l e and r e s e l e c t e d runs. The s i n g l e s e l e c t i o n runs (10a, lOd, and lOe) were  summed to give classes of putative mutant, other survivors, and dead. The reselection runs (10b and 10c) were likewise grouped. The difference was s i g n i f i c a n t at the 99*5% certainty l e v e l .  43 BIBLIOGRAPHY Adoutte, A., and J. Beisson. (l970). Cytoplasmic inheritance of erythromycin resistant mutations i n Paramecium aurelia. Molec. Gen. Genetics 108j 70-77. . (1972). Evolution of mixed populations of genetically different mitochondria in Paramecium aurelia. Nature 235* 393~396. Allen, S. L. (1967). Cytogenetics of Genomic Exclusion i n Tetrahymena. Genetics 5J.1 797-822. Beale, G. H. (1969). A note on the inheritance or erythromycin resistance in Paramecium aurelia. Genet. Res. 14: 341.  /  Beale, G. H., Knowles, J. K. C., and A. Tait. (1972). ' genetics in Paramecium. Nature 235: 396-397.  Mitochondrial  Berger, J. D. (1971)• Kinetics of incorporation of DNA precursors from ingested bacteria into macronuclear DNA in Paramecium aurelia. J . Protozool. 181 419-429. ; (1973). Selective inhibition of DNA synthesis in macronuclear fragments i n Paramecium aurelia exconjugants and i t s reversal during macronuclear regeneration. Chromosoma (Berl.) in press. Berger, J. D., and R. F. Kimball. (1964). Specific Incorporation of Precursors into DNA by Feeding Labelled Bacteria to Paramecium aurelia. J. Protozool. 11: 534-537. Boyce, R., and R. Setlow. (1963). The Action Spectra for UltravioletLight Inactivation of Systems Containing 5-Bromouracil Substituted Deoxyribonucleic Acid. Biochim. Biophys. Acta. 68: 446-454. Butzel, H. M., Jr. (1953). The genetic basis of mating type determination and development in the varieties of Paramecium aurelia belonging to Group A. Doctoral dissertation. Indiana University. Bloomington. Byrne, B. C. (1973). Mutational analysis of mating type inheritance in syngen 4 of Paramecium aurelia. Genetics 7_4s 63-8O. Cerda-Olmedo, E., and P. C. Hanawalt. (1967). Macronuclear Action of Nitrosoguanidine in Escherichia c o l i . Biochim. Biophys. Acta. 142: 450-464.  Cerda-Olmedo, E., Hanawalt, P. C., and N. Guerola. (1968). Mutagenesis of the replication point by nitrosoguanidine: map and pattern of replication of the Escherichia c o l i chromosome. J. Mol. Biol. jQ*  705-719.  - Cleffman, G. (1968). Regulierung Der DNS-Menge in Makronucleus von Tetrahymena. Exp. Cell Res. 5.0* 193-207. Cohen, S. S., and H. D. Barner. (1954).. Studies in Unbalanced Growth in Escherichia c o l i . Proc.Natl.Acad.Sci.U.S. 44: 1004-1012. * Benhur, E. and M. M. Elkind. (1972-). Damage and Repair of DNA in 5-Bromodeoxyuridine-labelled Chinese Hamster cells exposed to fluorescent light. Biophysical Journal, 12,: 636.  Cohen, S. S>, Flacks, J. G . . Barner, H . D., Loeb, M. R,, and J. Lichtenstein. (1958)• The Mode of Action of 5-Fluorouracil and i t s Derivatives. Proc.Natl.Acad.Sci.U.S. 44: 1004-1012. Cook, J . R., and T. W. James. (1964). Age distribution of cells i n logarithmically growing c e l l populations. In Synchrony in c e l l division and growth, pp 485-495* E. Zeuthen (ed.) New Yorkj Inter science Publishers, Inc. Dippell, R. (1955)• A temporary stain for Paramecium and other c i l i a t e protozoa. Stain Tech. J D : 69-71. Dodson, M. L., Hewitt, R. and M. Mandel. (1972). The nature of UV light induced strand breakage i n DNA containing Bromouracil. Photochem. & Photobiol. 16: 15-25. f  Donachie, W. D. (1969). Control of c e l l division in Escherichia c o l i . J. Bact. 100: 260-268. Donachie, W. D., and D. G. Hobbs. (1967)« Recovery from "Thyraineless Death" in Escherichia coli 15T~. Biochem. Biophys. Res. Commun. 22: 172-177• Driver, E. C. (1931). J. Exp. Zool. 5_£:  Temperature and Gene Expression in Drosophila. 1-28.  Dryl, S. (1959). Antigenic Transformation i n Paramecium aurelia After Homologous Antiserum Treatment During Autogamy and Conjugation. J. Protozool. Suppl. 61 25. Edgar, R. S., and I. Lielausis. (1964). Temperature-sensitive Mutants of Bacteriophage T^D: Their Isolation and Genetic Characterization. Genetics 42: 649-662. Epstein, R. H., Bolle, A., Steinberg, C. M., Kellehberger, E,, Boy Dela Tour, E., Chevalley, R., Edgar, R. S., Susman, M,, Denhardt, G. H., and A. Lielausis. (1963)* Physiological Studies of Conditional Lethal Mutants of Bacteriophage T D, Cold Spr. Harb. Quant. Biol. 28: 375-394. Frankel, J. (1967). Studies on the Maintenance of Oral Developments i n Tetrahymena pyriformis GL-CII. The Relationship of Protein Synthesis to Cell Division and Oral Organelle Development. J. Cell Biol. 34:  841-858.  Goldin, A., Venditti, J. M., and I. Kline. (1959). Evaluation of Anti-leukemic Agents Employing Advanced Leukemia L-1210 i n Mice. Screening Data Cancer Res. 19_: 429-466. Greene, M. 0.,aand J. Greenberg. (i960). The Activity of Nitrosoguanidines Against Ascites Tumors i n Mice. Cancer Res. 20: 1166-1171. Greer, S. and S. Zamenhof. (1957). Effect of 5-Bromouracil i n Deoxyribonucleic Acid of E.coli on Sensitivity of Ultraviolet Irradiation. Am. Chem. Soc. Abstr. 131st Meeting, p. 3c.  45 Gross, J# D. (1972). DNA replication i n bacteria. Immun. 5_7_» 39-74.  Curr. Top. Microbiol.  Guerola, N., Ingrahafll, J . L., and E. Cerda-Olmedo. ( l 9 7 l ) t Induction of closely linked multiple mutations by nitrosoguanidine. Nature New Biol. 220t 122-125. Hartwell, L. H. (1967). Macromolecule Synthesis in Temperature Sensitive Mutants of Yeast. J . Bacteriol. 23_: 1662-1670. Helmstetter, C. E., and 6 . Pierucci. ( 1 9 6 8 ) . Cell Division During Inhibition of Deoxyribonucleic Acid Synthesis i n E.coli. J. Bact. 95?  1627-1633.  Hjelm, K. K. and E. Zeuthen. (1967). Synchronous DNA Synthesis Induced by Synchronous Cell Division in Tetrahymena. •CR. Trav. Lab. Carlsberg 36: 127-160. Hoffman, J. G. (1949). Theory of the Mitotic index and i t s application to Tissue Growth Measurement. Bull. math. Biophys. I l l 139-144. Hotchkiss, R. D. (1954). Cyclic Behaviour i n Pneumococcal Growth and Transformability Occasioned by Environmental Changes. Proc. Natl. Acad. Sci. U.S. 40: 49-55. Humphrey, R. M., Steward, D. L., and B. A. Sedita. (1969). DNA Strand Scission and Rejoining Mammalian Cells, i n Genetic Concepts and Neoplasia, p. 570. Williams and Wilkins, New York. Hutchinson, F., and W. Koehnlein. (1967). The Mechanism by which Bromouracil Sensitizes DNA to Ultraviolet. Radiation Res. Abstr. J l : 547. Kimball, R, F. ( l 9 6 l ) . Post-irradiation processes i n the induction of recessive lethals by ionizing radiation. J. Cell and Comp. Physiol. 581  163-170.  . (1963). The relation of repair to differential radiosensitivity in the production of mutations i n Paramecium. In Repair from genetic radiation damage and d i f f e r e n t i a l radiosensitivity in germ cells. F. H. Sobels (ed.) Pergamon Press, New York, pp. I 6 7 - I 7 8 . Klein, A., and F. Bonhoeffer. Biochem. 41: 3OI-332.  (1972).  DNA replication. Ann.  Rev.  Kung, C ( l 9 7 l ) . Genie mutants with altered systems of excitation i n Paramecium aurelia I I : Mutagenesis, screening and genetic analysis of the mutants. Genetics. 6_9_: 29-45. Ley, R. D., and R. B. Setlow. (1972). Rapid Repair or Lesions Induced by 313 nm. Light i n Bromouracil-Substituted DNA of Escherichia c o l i . Biochem. Biophys. Res. Commun. 46: 1089-1094. Leiter, J . , and M. A. Schneidernann. (1959). Screening Data from the Cancer Chemotherapy National Service Center Screening Laboratories. Cancer Res., 19_: 31.  46  Loomis, W. F., J r . (1969). Temperature-sensitive Mutants o f D i c t y o s t e l i u m discoideum. J , B a c t e r i o l . .9_9j 65-69. Menningman, H. D., and W, S z y b a l s k i . (1962). Molecular Mechanism o f Thymineless-Death. Biochem. Biophys. Res. Comsun. 9_i 398-404. Mitchison, J, M. ( l 9 7 l ) . Press. Cambridge.  The biology o f the c e l l c y c l e .  Cambridge Univ.  Neidhardt, F. C. (1964). The Regulation o f RNA Synthesis i n B a c t e r i a , i n J . N. Davidson and Waldo E. Conn (ed.), Progress i n n u c l e i c a c i d research and molecular b i o l o g y , pp. 145-181. V o l . 3« Academic Press, Inc. New York. Orias, E., and M. F l a c k s . (1973). Use o f Genomic "Exclusion t o I s o l a t e Heat-sensitive Mutants i n Tetrahymena. G e n e t i c s , 23.' 543 559« _  Orias, E., and N. A. P o l l o c k . (1973)• Temperature-sensitive phagocytosis i n a Tetrahymena mutant. Progress i n Protozoology. Abstracts o f papers read a t the IV I n t . Cong. Protozoology. Clemont-Ferrand 2-9 Sept. 1973. P a i n t e r , P. R., and A. G. Marr. (1968). populations. A. Rev. M i c r o b i o l . 22:  Mathematics o f m i c r o b i a l 519-548.  Pasternak, J . (1967). D i f f e r e n t i a l Gene A c t i v i t y i n Paramecium a u r e l i a . J . Exp. Z o o l . I65.1 395-418. Pauling, C., and L. Hamm. (1968). P r o p e r t i e s o f a Temperature-Sensitive Radiation - S e n s i t i v e Mutant o f E s c h e r i c h i a c o l l . Proc. N a t l . Acad. S c i . U.S. 60: 1495-1502. P i e r u c c i , 0. (1969). Regulation o f C e l l D i v i s i o n i n E s c h e r i c h i a c o l i . Biophys. J . 9_i 90-112. P i n t e r , K. G., Hamilton, J . G., and 0. N. M i l l e r . (1963). L i q u i d S c i n t i l l a t i o n Counting w i t h Glass F i b e r Paper. Anal. Biochem. 5_" 458-463.  458-463.  P o l l o c k , S. (1970). Studies on the g e n e t i c s and development o f t r i c h o c y s t s i n Paramecium a u r e l i a syngen 4. Ph.D. t h e s i s . Indiana U n i v e r s i t y , Order No. 71-19271 U n i v e r s i t y M i c r o f i l m s , Ann.Arbor, Mich. Puck, T. T., and F. T. Kao. (1967). Genetics o f Somatic Mammalian C e l l s , V. Treatment with 5-bromodeoxyuridine and v i s i b l e l i g h t f o r i s o l a t i o n o f N u t r i t i o n a l l y D e f i c i e n t Mutants. Eroc. N a t l . Acad. S c i . U.S. 58: 1227-1234. Rapaport, S. A. (1964), A c t i o n Spectrum f o r I n a c t i v a t i o n by U l t r a v i o l e t L i g h t o f Bacteriophage Th S u b s t i t u t e d w i t h 5-B"romodeoxyuridine, Virology 22: 125-130. Rodriguez, R. L,, Dalbey, M. S., and C. I . Davern. (1973)• Autoradiography Autoradiographical Evidence f o r B i d i r e c t i o n a l DNA R e p l i c a t i o n i n E s c h e r i c h i a c o l i . J . Mol. B i o l . 7j£: 599-604.  47  Roberts, C. T., and E. O r i a s . (l973)» Cytoplasmic i n h e r i t a n c e of chloramphenicol r e s i s t a n c e i n Tetrahymena. Genetics 7_3_: 259-272. Scherbaum, 0., and E. Zeuthen. (1954). Induction of Synchronous C e l l D i v i s i o n i n Mass Cultures of Tetrahymena p y r i f o r m i s . Exp. C e l l Res.  6:  221-227.  Schubach, ¥. M., and C. I . Davern. (1971). Biophys. Soc. Abstr. i n Biophys. J . 235a. ( u n a v a i l a b l e , c i t e d i n Rodriguez, Dalbey and Davern. 1973) Seyster, E. W. (1919). Eye Facet Number as Influenced by Temperature i n the Bar Eyed Mutant of Drosophila. B i o l . B u l l . %?i 168-181. Skinner, W. A., Gram, H. F., Greene, M. 0., Greeriberg, J . , and B. R. Baker. (i960). Potent Anticancer Agents-XXXI. The R e l a t i o n s h i p of Chemical Structure to Antileukaemic A c t i v i t y with Analogues of l - M e t h y l - 3 - N i t r o - l - N i t r o s o g u a n i d i n e . J . Med. Pharm. Chem. 2: 299-333. Smets, Lo A., and J . J . C o r n e l i s . (l97l)« Damage i n 5"Bromouracil S u b s t i t u t e d DNA I n t . J . Radiat. B i o l , l ^ t 445-457.  Repairable and I r r e p a i r a b l e Exposed to U l t r a - v i o l e t Radiation.  Smith, C. L., and P. P. Dendy. (1962). R e l a t i o n between m i t o t i c index, duration of m i t o s i s , generation time and f r a c t i o n of d i v i d i n g c e l l s i n a p o p u l a t i o n . Nature, Lond. 193:  555"556,  Sonneborn, T. M. (1965). Degeneracy o f the Genetic Code: Extent, Nature, arid Genetic I m p l i c a t i o n s i n E v o l v i n g Genes and P r o t e i n s . V. Bryson and H. Vogel (ed.) Acad. Press, Inc. New York. _______ (1970). Methods i n Paramecium Research, i n Methods i n C e l l P h y s i c s , v o l . 4. D. M. P r e s c o t t (ed.) Academic Press, Inc. New York. S t a h l , F. W., Crasemann, J . M., Okun, L., Fox, E., and C. L a i r d . (1961). Radiation S e n s i t i v i t y of Bacteriophage Containing 5~Bromodeoxyuridine. V i r o l o g y . 13.:. 98-104. Stanners, C. P., and J . E. T i l l . (i960). DNA synthesis i n i n d i v i d u a l L - s t r a i n mouse c e l l s . Biochim. Biophys. Acta. 3JZ.:. 406-419. S t e i s i n g e r , G., Muai, F., Dreyer, W. J . , M i l l e r , B., and S. H o r i u c h i . ( l 9 6 l ) . Mutations A f f e c t i n g the Lysozyme o f Phage T^. Cold Spr. Harb. Symp. Quant. B i o l . 26: 25-30. Suzuki, D. T. (1970). Temperature-Sensitive Mutations i n Drosophila melanogaster. Science 170: 695-706. Taub, S. R. (1966). U n i d i r e c t i o n a l mating type changes i n i n d i v i d u a l c e l l s from s e l f i n g c u l t u r e s of Paramecium a u r e l i a . J . Exp. Z o o l . 163: 141-150.  *  Unrau, P., and R. H o l l i d a y . (1970). A search f o r temperature-sensitive mutants of U s t i l a g o maydis blocked i n DNA s y n t h e s i s . Genet. Res., Camb. 15_: 157-169.  *  Taylor, M. ¥., Spuhrada, M., and J . McCall. (l97l). Mutants of Chinese Hamster c e l l s . Science. 172:  New Class of Purine 162-163.  48 Wacker, A., Dellweg, H., and Weinblum, D. (l96l). Uber d i e Strahlensen s i b i l i s i e r e n d e Wirkung des 5 Bromuracils. J . Mol. B i o l . 3j 787-789. W h i t f i e l d , H., Martin, R., and B. Ames. (1966). C l a s s i f i c a t i o n o f Amino t r a n s f e r a s e (C Gene) Mutants i n t h e H i s t i d i n e Operon. J . Mol. B i o l .  211  335-355-  Woodward, J . , Belber, B., and H. S w i f t . ( l 9 6 l ) . Nucleoprotein changes during the m i t o t i c c y c l e i n Paramecium a u r e l i a . Exp. C e l l Res. 23?  258-264.  49  APPENDIX I GROWTH CURVES FOR ESCHERICHIA COLI STRAINS USED Growth Curves f o r 15TAU" E s c h e r i c h i a c o l i @ 37° C In the growth curves, A was supplemented with Bacto-casitone, while B and C had Casamino a c i d s . (2.5mg/ml.) which might l a c k some metabolite which was present i n casitone; thus the increased lag would have occurred while there was i n d u c t i o n of b i o s y n t h e t i c pathways to make the product. The c u l t u r e s were a l s o supplemented w i t h thymine (30 g/ml.), u r a c i l (15 g/ml.) and dextrose (5mg/ml.). In the s e l e c t i o n s , 24 g/ml. of 5-bromouracil was s u b s t i t u t e d f o r thymine. In a 10ml. c u l t u r e , of c u l t u r e , the proportions were 9ml. stock P i e r u c c i ' s modified M-9 s a l t s , 50mg. dextrose, 25mg. casamino a c i d s , 150 g. u r a c i l , and J00 g. thymine o r 240 g. of 5-bromouracil. 15TAU~ was used i n s e l e c t i o n s 4b, 5a, 5b, 6a, and 6b (Table I V ) . The s t r a i n was a l s o u t i l i z e d i n the f i r s t 3H-thymidine uptake t e s t s on p u t a t i v e t s l i n e s (except f o r #63 and #125) i n Table Va, p a r t 1 of the Results. Growth Curves f o r 15T~ E s c h e r i c h i a c o l i @ 37°C A, B, and C were three separate c u l t u r e s a l l e x h i b i t i n g lengthy lag (245-3?5 minutes) before growth. The c u l t u r e s were supplemented as w i t h 15TAU". . 15T" was used i n s e l e c t i o n s 7a, 7b, 7c, 7d, and 7e (Table IV) as w e l l as i n the vegetative dosage and clone forming a b i l i t y experiments. Section I I I of the R e s u l t s . Growth Curves f o r 15T~55-7 E s c h e r i c h i a c o l i @ 35°C Growth was s i m i l a r on thymine (30 g/ml.) and on 5-bromouracil (24 g/ml.). There was no a g i t a t i o n during these experiments. T h i s s t r a i n was supplemented as with the other two. I t proved to be the best of the three, being r e s i s t a n t to long cold storage a t 4°C, and e a s i l y r e i s o l a t e d from contaminated c u l t u r e s . 15T~55"7 was used i n s e l e c t i o n s 10a, 10b, 10c, lOd, and lOe (Table I V ) , as w e l l as i n the Paramecium ^H-BU uptake experiment (Table I I of the M a t e r i a l s and Methods), the Marked-Recapture experiment (Section I I of the R e s u l t s ) , and the 3H-thymine t e s t of DNA i n c o r p o r a t i o n on p u t a t i v e t s l i n e s (Table Va, p a r t s 2 and 3 of the R e s u l t s ) .  50  APPENDIX  I  (growth  curves)  /  51  APPENDIX  i: (growth  curves)  0.5-  OA-  / S X  A / / B  J C  /  h  o.3-  . E.coli  15T  S J  "^0.2 •  o  /  J  x  /  J  I  «5  J05  565  425  455  545  J  605  ' 665  ' 725  MINUTES  Q5-  °- '  thymine  4  Ecoli  15T~55-7  .  ^x'  X* ^^-°^°"~ " cr  n  ^°~°^°  5-bromouracil.  x-—x^  0  ' 6 0  120 MINUTES  180  240  52 APPENDIX I I T h e o r e t i c a l Scheme f o r the Use o f t s DNA Mutants i n an Experiment Concerning t h e Control o f DNA Metabolism The i n i t i a l cross o f mutant by w i l d type simply y i e l d s heterozygotes which would be backcrossed to the mutant, g i v i n g an F£ w i t h two c l a s s e s o f c e l l s i n each mating type. One c l a s s of c e l l s ( c y t o p l a s m i c a l l y d e r i v e d from t h e F T heterozygote) would have the genotype t s / t s , t s / t s , and ts/+ f o r the macronuclei, m i c r o n u c l e i , and macronuclear fragments r e s p e c t i v e l y . Now i f t h i s c e l l was subjected to the r e s t r i c t i v e temperature (to express the mutant phenotype), both, n u c l e i could not synthesize DNA. T h i s s i t u a t i o n could be remedied i f the macronuclear fragments (genotype ts/+) "switched on" synthesis of DNA. T h i s may f o r c e the c e l l t o go through a forced macronuclear regeneration a phenomenon only r a r e l y seen i n the l i f e c y c l e .  

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