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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 TEMPERATURE-SENSITIVE 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 t h i s t h e s i s i n p a r t i a l f u l f i l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h C olumbia, I agree t h a t the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . 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 e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y p urposes may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l n o t be a l l o w e d w i t h o u t my w r i t t e n p e r m i s s i o n . Department The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada (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 in 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 ( i i ) (Equilibration time before bromouracil treatment) have been optimized. The relative effects of parts of the screening procedure are presented along with discussion of the results, and suggestions for future appli-cations of the isolated Paramecium lines. ( i i i ) TABLE OF CONTENTS PAGE L i s t of Tables . . . . . . . . . . . . . . . . . i v L i s t of Figures v Acknowledgement . . . . . . . . . . . . . . . . . . . . • v i Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Materials and Methods . . . . . . . . . . . . . . . 5 Results I Y i e l d of the Selection System on Mutagenized Populations . . . . . . . . . . . . . 15 . I I E f f i c i e n c y of the Selection System (Marked-Recapture Experiment) . . . . . . . . . . . . . . . 25 I I I Pattern of Decrease i n Survival and Clone Forming A b i l i t y Due to Selection . . . . . 26 IV Relative Contribution of Components of the Selection System to Total C e l l Death . . . . . . . . . . . . 28 Discussion Mutagenesis and the Selection System . . . . . . . . 32 Rationale f o r the Selection System and Possible Applications . . . . . . . . . . . . . . . . . . . 37 Concluding Statement . . . . . . . . . . . . . . . . . . . . . 39 Notes . 40 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Appendices I Growth Curves f o r Escherichia c o l i Used . . . . . . . . . o . . 49 I I Theoretical Scheme f o r the Use of ts'DNA Deficient Mutants i n an Experiment Concerning the Control of DNA Metabolism . . . . . . . . . . 52 (iv) LIST OF TABLES PAGE I Isotopes Used . . . . . . . . . . • 7 II Incorporation of DNA Precursors from Labelled Bacteria into Paramecium aurelia at 23 and 35°C 9 III Fate of Mass Cultures During the Mutagenesis Treatments . • • . • • • • • • * • • » . « . . . H IV Yield of the Selection System 15 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 Approximate ^H-Thymine Incorporation . . . . . . . . . . . . 20 Part 3 Quantitative ^H-Thymine Incorporation . . . . . . . . . . . . . . . . . . 21 b Protein Synthesis at 27 and 37°C 22 c Food-vacuole Formation at 23 and 35°C . . . . . . . . . . . 23 d Growth at 27°C . . . . . . . . . . . . . . . . 24 VI Efficiency of the Selection System (Marked-Recapture Experiment) 25 VII Relative Contribution of Components of the Selection System to Total Cell Death . 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 . . . . . . . . . . . . . . . . 2? B Clone Survival with Varying Light Exposure . . . . . . . . 27 5 Effect of Light Exposure and Delay Before Isolation on Clone Survival . . . . . . . . . . . . . . . . 31 (vi) ACKNOWLEDGMENT I wish to 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. Also, 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 disaster and f a i l u r e . The technical assistance and constructive c r i t i c i s m given by E r i c was especially appreciated. This research was supported by N.R.C. operating grant A-63OO to Dr. J . D. Berger. INTRODUCTION Conditional mutations provide one genetic means to examine essential functions i n any prokaryotic or eukaryotic organism. The Bar locus i n Drosophila melanogaster was the f i r s t gene shown to have variable expression as a function of temperature (Seyster, 1919; Driver, 1931)• However, conditional mutations of t r u l y indispensible functions were not exploited u n t i l S t r e i s i n g e r et a l . . (1961) i s o l a t e d temperature sensitive (ts) mutants of lysozyme i n Escherichia c o l i . Epstein 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 controls of es s e n t i a l metabolic and deveXopsental processes. Since that time t s mutations have turned out to be an increasingly important genetic t o o l . Ts mutants have been used to study macromolecular synthesis i n Escherichia c o l i (Neidhart, 1964) ao£ i n Saccharomyces cerevisiae (Hartwell, 1967). This class of mutations', has also been used to probe development i n Dictyostelium  discoideam (Loomis, 1969) ajid i n Drosophila melanogaster (Suzuki, 1970). WA synthesis i s an essential function which has been vigorously explored g e n e t i c a l l y through use of conditional mutants i n prokaryotes (Klein and Bonhoeffer, 1972; Gross, 1972). However, f a r l e s s work has been done on the genetic basis of DNA r e p l i c a t i o n i n eukaocyotes (Hartwell, 1967; Unrau and Holliday, 1970). Such studies have Jbeeft hampered by the lack of a preliminary screening procedure which could be applied to mass cultures; most of the mutants have been recovered! by r e p l i c a - p l a t i n g of i s o l a t e s at the permissive and r e s t r i c t i v e temperatures. The goal of t h i s study was to devise a preliminary screening procedure, applicable to mass cultures of Paraiaeciga a n r e l i a . which would allow s e l e c t i o n of t s DNA d e f i c i e n t 2 mutants. The screening procedure is 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, light has l i t t l e or no effect on DNA unless i t is BU•substituted (Dodson, Hewitt, and Mandel, 1972). Using BU and radioactive phosphate to label A phage DNA, they demonstrated a linear release of phosphate in BU substituted cultures and no release in 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 in 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 in 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 killed by exposure to 313 nm. light 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 in tissue culture cells using BU photolysis. . UV photolysis of BU substituted DNA (Boyce and Setlow, 1963) has been used in radiation sensitivity assays in Escherichia coli (Greer and Zamenhof, 1957) and bacteriophage T^ (Stahl et al . , 1961). It has also been used, in Escherichia coli to recover ligase mutants (Pauling and Hamm, 1968) and in 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 lethality was not expected because the chance of damaging a l l 800 copies of a gene responsible for a particular essential function i n the macronucleus seemed small. This fortunate vegetative death made selection of large numbers of ce l l s possible; without i t , only relatively small numbers of cell s could have been screened since individual c e l l lines would have had to be followed through the following autogamy (position 2). Overall, when both types of death are summed, the Selection System enriches for a ts mutant \ obtained through the screen, by approximately 7500 fold 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 for 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 It was hoped that the project would produce a selection system for ts DNA deficient cells that was practical enough for 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 definition of the screening procedure design and has l e f t the generation and analysis of large numbers of mutants to others. figure 1 SELECTION SYSTEM A 35K F E E D Equilibration BACTERIA °y 27 t NG o \ Expression /17 \ Autogamy + 6hrs 6hrs f 1hr DARK Autogamy 1/ Feed Test IRRADIATE position 2 12 hrs position 1 Start t -NG Expres- Veg sion j j death 1 DAY — I — I — 7 8 Auto-gamy + 11 1213 15 Selection 35°C + Exautogamous death | ^ 18 Autogamy 22 24 27 Testing MATERIALS AND METHODS 5 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 plastic 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 for v i a b i l i t y according to Kimball (1963)» "very poor" was equivalent to one fi 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 fissions; "bad", three fissions; and "good", four or more fissions. These groups were lumped into "subviable", zero to two fissions, and "viable", three or 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 restrictive and permissive temperature, (Table Va, part l,and Table Vb). In the second part of Table Va, a rough estimate of this ratio was made -using a 5 category ranking scale; then promising lines 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 direct count of vacuoles containing carbon particle 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) in 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 coli 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 for these strains can be found in Appendix I. Production of BU substituted Escherichia coli 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 in modified M-9 salts three times. The pellet was resuspended in 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 in Dryl's salt solution (Dryl, 1959) ready to be fed to the paramecia. Isotope 3H-thymidine (methyl-^H) ^H-thymine (methyl-^R") •^H-5-bromouracil (C6-\) ^H-leucine (C4 ,5-^H) TABLE I ISOTOPES USED Spec. Act. (c/mmol.) 5.0 1.9 8.7 31.9 Source Amersham Schwartz Mann Schwartz Mann New England Nuclear Tritium labelled BU (Table i) was used to measure uptake into 15T 55"7 Escherichia coli at 35°C. Label was taken up at an even rate as the culture grew (Figure 2). 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 in a one percent solution of BU and air dried). The spotted f i l t e r s were immediately immersed in five percent trichloroacetic acid with five percent BU and kept on ice for at least three hours. Three successive washes in the same but fresh solution followed by alcohol-ethyl ether (lsi) and ethyl ether finished the precipitation. The discs were then air dried at 80°C and placed into liquid scintillation vials, spot side up. The precipitated acid insoluble material was then ready for counting. Cell number was not estimated directly by turbidity (optical density); but by clone forming ability 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 in both thymine and bromouracil supplemented cultures. If the bacteria should run short of DNA precursor before stationary phase 8 is reached, the cells would exhibit the elongation ("chain" or "snake") effect well known in Escherichia coli undergoing thymineless death or related metabolic crises (Cohen and Bamer, 195^ } Cohen et al . , 1958; Menningman and Szybalski, 1962). In other words, Figure 2 is based on actual cell numbers (Figure 3 corrected) which cannot be directly estimated by turbidity. The counting efficiency was higher with glass fil t r e s (GF/A) as shown by Pinter, Hamilton, and Miller (1963). FIGURE 2 5-BR0M0URACIL INCORPORATION BACTERIA AT 36°C FIGURE 3 BACTERIAL COLONY FORMATION VERSUS TURBIDITY H-12-§ 7 0 -' S8 -GF/A / 3 M M r "T 0 60 120 180 M I N U T E S 240 w1 0 0.1 0.2 0.3 OPTICAL 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 in unlabelled medium, dried on albumenized slides, Feulgen stained, and autoradiographed. The results (Table II), show that incorporation is not significantly lower at the 9 higher temperature. The experiment indicated that BU entered the macronucleus of cells in 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 0.66 23°C 35°c 35 39 21.2 + 1.8 17.8 ± 1.4 0.62 *Sample sizes varied because some cells were in autogamy. Only cells with vegetative nuclei were scored. Liquid Scintillation Counting Liquid scintillation 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) in 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 in a single microdrop and allowed to dry. After fixation in Carnoy's (ethanolacetic, 3«l) for 20 minutes, the slides were hydrolysed in 1 Normal HC1 at 60°C for 10 minutes, and Feulgen stained for 60 minutes. Liquid nuclear track emulsion (illford K-5) was applied as a $Q>% w/w solution with water at 70°C. After the slides were dipped in emulsion, they were dried in 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 for tritium label, i t was imperative that the nuclear a c t i v i t y be estimated autoradiogra-phically since both Paramecium DNA and bacterial DNA (caught in 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 ce l l s were harvested by negative geotaxis (they congregate at the neck of a four l i t e r erlenmeyer flask, where they are easily siphoned off) 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. for 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 after autogamy when a ratio of dead to i n i t i a l number was calculated. To test exautogamous l e t h a l i t y , and to check for losses of cells 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 in cell number. However in Treatment B, the cells doubled in number indicating that active metabolism Post Auto-gamous Loss 43.5# 40.US 3*1% 64.8?S 61.2% 3.6% occurred during the exposure to nitrosoguanidine. Again, there was a loss due to the toxicity of NG (23.9%)• 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 in 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 is five to 10 fissions). A sample of 5 x 10^ to 1 x 10^ cells was heated to 3^«5°C for six hours in a water bath and then BU substituted bacteria (prewarmed to 3^»5°C) were fed to the animals for six hours in darkness. This was followed by quickly but gently washing the cells by centrifugation. They were then resuspended in 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. TABLE III FATE OF MASS CULTURES DURING THE MUTAGENESIS TREATMENTS Mutagen Treat. A Exptl. Cont. B Exptl. Cont. Pretreat. Number 4.0xl0c 2.6x10* 1.4xl0c 3.4x10* Post Treat. Number 2.7x10^ 2.0x10* 3.0x10" 8.1x10* Loss* NG+ Loss Handling NG -32.5^ -23. UK 114.3# 138-.2J6 9.4^ 23.9^ 12 7 The c e l l s were p e r i o d i c a l l y checked f o r body deformation and l y s i s . An a d d i t i o n a l Dryl's wash several hours i n t o the l i g h t exposure was done i f large amounts of debris were present i n the cu l t u r e . A f t e r 12 hours of 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 with culture f l u i d and stored at 17°C or 27°C. Animals showing any signs of swelling e a r l y i n the l i g h t exposure (four to s i x hours) di e d soon a f t e r i s o l a t i o n . Consequently, the h e a l t h i e s t looking specimens were rescued f i r s t and then a small amount of c u l t u r e f l u i d was added so that " l o s t " survivors had an opportunity to feed on b a c t e r i a rather than the dismembered bodies of t h e i r l y s e d peers. Paramecia w i l l r e a d i l y consume material from l y s e d c e l l s . In s e l e c t i o n "runs, t h i s probably includes f r e e BU l i b e r a t e d by l y s i s of the majority of c e l l s . A l l c e l l s (including mutants) would therefore be vulnerable to UV l i g h t damage as a r e s u l t of t h i s secondary uptake. Thus the optional Dryl's wash was often used (the animals were allowed to swim up from the 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 re-exposed). 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 recovered from the screening procedure were c a l l e d "survivors". 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 to one f i s s i o n a t the r e s t r i c t i v e temperature a f t e r 2M- hours, and three to f i v e f i s s i o n s at the permissive temperature) were selected from these survi v o r s . At l e a s t two more t s t e s t s were conducted before the l i n e s were examined autoradiographically f o r DNA incorporation. Wild type c e l l s r o u t i n e l y grew at a r a t e of f o u r to f i v e f i s s i o n s per day a t 27°C; at 36°C, wild type c e l l s survived but d i d not d i v i d e . At 3^ to 35°C the c e l l s grew at a meexi r a t e of k.k3 d i v i s i o n s per day (Table V of Results). 34.5°C was chosen therefore as the r e s t r i c t i v e temperature. T h i s !3 i s in disagreement with Sonneborn (1970) who states that 35 to is "an excellent 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 until 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 air incubator to heat the animals to the restrictive temperature, there is reason for allowing a putative ts line in 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 in a water bath, exposing individual dividers in 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 ts tests are in 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 of the Selection System on Mutagenized Populations A summary of a l l selection runs i s shown i n Table IV. The length of the irradiation exposure was varied considerably u n t i l approximately 12 hours was adopted exclusively (from experiment 7e on). Of the five 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 + + VO O N O O H ^ H O N N N V ^ ^ - J -i-H CM UN C 0 H \ O » A ( M N rH CM rH CM H|<M 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 i-)|o)nf*-nW-inLfp.|4-\QVOVO O- C~\ CM H O v O O- vj^  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>- CNOO CM -3" ON mo 4- ON CM ON CM 00 ON CO C^VO C\! \Q H 3^" C\! VO ! !0 >0 S *0 "*> rH rH rH rH rH I t>CNSCMC\l<NlC\JC\tvOvO\OvOvO VO O-„ * „ O O O CO • • ON rH VO O I x> cd x» B J d ^ O T ) 0 ) d f l O ' d ( i ) <r U N V O o-o-c^o-o-ooooo H H r i r i H -P 10 CD 4-> e S - P an «H tO 0 © JQ U •rt cd o rH we -P to 00 H CO a o 3 cd rH CO £ © xi a -3- • cd to rH o £ rH CO XI o O rH • xi CO CO © cd xi H -P vH O © cd c rH c CO c •v. X to s CO e bp to U ? -H £ © ft <u -p ru -P ft ru © c X O © •rt CO •P •P 10 -p o O -H © o s © (0 ft CO rH 0) (1) C -P © * 10 CO ! CO CO -ch eg rH H rH -PU • s o to • c -p > rH O © c M 3 rH © CH e £> CO CO CO •p rH CO -p o cd to xi © cd o >> u EH O to © -p (0 CO © © ur XI * rH ur •p O O C H o •rt o rH 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 fairly 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. Between one and 20 million 6 6 animals (1x10 to 2x10 being the most convenient) were treated in 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 itself. One of the successful test runs (4b) is included in Table IV. Out of 2x10^ cells, 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 cell per thousand. The f i r s t real test of the procedure came in 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 ts mutants). Two weeks later, the remaining mutagenized cells, which had been kept at 17°G in stationary phase, were selected with no survival. This was the f i r s t indication that the screening procedure was capable of kill i n g a l l cells in a run. Since the system could be harsh, mutagenized cells 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^ cells survived). During the irradiation, two classes of cells were seen (swollen cells and normal cells) which must 17 have been caused by abnormalities in 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 bacteria fed to the paramecia before irradiation (this was not the case in run 6a as strong turbidity was noted in the culture due to excess bacteria), or slow-growth of animals before the bacterial f eeds In the last case animals may have not formed a.s many food-vacuoles as usual. The classes (one badly damaged, the other undamaged) may have been caused by a threshold of lig h t damage to BU substituted DMA in the polyploid macronucleus. By this point in the study i t was obvious that standardization of the parameters controlling the screening system had to be stringent. Standardized culturing of BU labelled bacteria was carefully followed (see Materials, and Methods). Other parameters of the system, however were adopted more or less a r b i t r a r i l y , and were allowed variation; for example, the equilibration time (the period that animals were heated to 3^»5°C before feeding of substituted bacteria), a,nd the duration of exposure to BU substituted bacteria. An equilibration time equal to one c e l l cycle (six to eight hours) was used because any block i n either maintenance or i n i t i a t i o n of DNA synthesis should have been encountered during that time at the restrictive temperature. For example, an i n i t i a t i o n mutant i n S when the temperature shift occurred, should continue i n i t s c e l l cycle u n t i l the next G-^ -S boundary before halting growth. The period of irradiation was reduced to about 12 hours following the experiments on the relative contributribution of vegetative and exautogamous le t h a l i t y on the overall selective lethality (total k i l l ) : the data w i l l be presented in Section III of the Results. On occasion, the cells were given time after irradiation in a non-nutrient medium before isolation. By leaving the animals in Dryl's solution overnight, many subviable individuals were eliminated. This 18 reduced the number of single isolations to be made. Generally, abnormal looking cells 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), line 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 itself 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 H O O O o - o i n o o o o o 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 OJ r \ l T | 4 O H o O N?\ • • • • • • • • • • • • o o o o o o o o o o o o H ^ C O V > O O O O N O N O O O O U ^ O O O O N O O U ^ 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 • • • • • • • • • • • • 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 • • • • • • • • • • • • COOHO\(MCONON\ONNO\ • • • • • • • • • • • • (^N O N 0 0\^ O O O O O +1 +1 -H -H +1 +«4^i^ +1+1+1+1 H H rH -5F -5 rH (3 - P CD 6 •> J3 >t ci •H S CO CD rH •2 p -p o M+> c°\ TH c o era o c c o o CD >>,H co x: co - P 0) >> (1) -I— o o 3 CD . o TH o •H - - P S e e •rH X) 5 o • H - P O o o c CO o CO s - P co to a o O H-> x: o co rH * CO rH a +> N [ S H ( ^ > ^ C O N O H r>0 C ( N i C V J v o v O v O v O O - C O C O O N O O rH O Line Median Rank # at 27°G Median Rank # at 35°G Description of Ratio 35/27°G Cont 1 5 4 low 66 5 1 low 85 4-5 2-3 low 90 4 2 low 101 „ 0-1 1-2 high 123 1-2 5 high 124* 4 0 v. low 125 4 4 equal 1* 3-4 0-1 v. low 24 4 3 low 32 3 2-3 low-equal 42 3-4 3 equal-low 55 4 3 low 102 0-1 0 equal-low 103 4 3 low 126 3-4 2 low 127 1 1 equal 128 2 0-1 low 132 1 0-1 low Cont 2 4 3 low Table Va, Part 2. ^H-Thymine incorporation was scored on a ranking scale to identify low incorporation at 35°C. The median category is 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 in 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 35°C I O - r H I • • O O O -P o-cvj ro «* 8 CD W S - P O . CM O r H VO O O VO • • • H H O O O UN CO o n e • H rt +> m 0) CV! £ 3 3 O * • O O O P ? O O O O O O • • • O O O • CD CO UN UN Ov f J + l • • • CVJ r H O +1 +1 +\ £ 0 & CO UNVO • « • 1 0 0 W WN O H CN C O ^ o CD CQ c c •H rt 4.1 S ( D T I g o & I O O evi a c o * CM C O O N • t> • -ff CO CM t! t i +' Ov O vO • • •> CV-3- -3" CO CO CVJ CD CD -P C CVJ O r H O -'H-leucine ^H-leuclne Line 2?°G Mean 3?°G Mean Incorp. 4 SE. Incorp. 4 SE. 22 21.4±1.5 18.440.9 27 18.6±1.1 > 18.1*L.l 51 18.4±1.1 17.8±1.0 63 16.4±1.3 18.6*L.2 65 18.3±1.4 17.2tL.2 66 16.3±1.0 9.2±1.2 68 17.340.9 23.5±1.1 77 17.8±0.9 17.8±1.2 80 11.3±1.0 15.5±1.2 85 17.9±1.6 9.5±1.1 90 14.84L.0 13.6±1.4 101 13.5*0.6 l?.8±l.i 125 16.84L.0 13.34L.0 Cont 1 32.0+2.4 19.141.2 Cont 2 19.441.4 13.2±0.8 Ratio of Ratio of V 27 & 37°G 27ts & 27wt Means Means 0.09 0.86 1.08 0.79 0.97 0.93 0.71 0.97 0.92 0.18 1.13 0.82 O.63 0.94 0.91 0.00 0.56 0.82 0.00 1.36 0.86 0.71 1.00 0.86 0.92 1.37 0.57 0.01 0.53 0.84 0.38 0.92 0.74 0.00 1.32 0.67 0.05 0.79 0.84 0.00 1 0.60 1.00 0.00 0.68 1.00 Table Vb. Incorporation of protein precursor at 27 and 37°G. A l l descriptions from Table Va, part 1 apply i n this table. Food-vac. Line 23°C Mean Form'n + SE. Food-vac. 350C Mean Form'n t SE. Ratio of 23 & 35°G Means Ratio of 27ts & 27wt Means 51 65 66 68 80 85 90 100 101 123 124 pwA Cont Line 63 125 1 24 32 103 126 127 128 132 7.7±0.7 5.5±0.7 9.0+0.9 9.3^0.7 8.6±0.6 7 . l t L . 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 Approx. Ratio of 23 to 35°G Means 1.50 1.00 0.50 1.00 1.00 0.50 0.50 0.66 1.00 0.00 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 . l i 0 . 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 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 Table Vc. Food-vacuole formation at 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 ratio of formation at high and low temperatures. These data were standardized against wild types which gave a ratio of Wo (35/23°G means). The second part of Table Vc shows approximate food-vacuole formations for some putative ts lines. **) o o V > a o *j S I s ro O O t-3 o N> Vol TABLE Vd PUTATIVE MUTANT GROUP ANALYSIS GROWTH AT 27°G (FRACTION OF CONTROLS) Line G r ° S o n a t 27° C 51 1.08 63 1.00 65 0.75 66 0.91 68 0o94 80 1.08 85 0.97 90 1.04 100 1.04 101 1.04 123 1.11 124 1.01 125 1.11 1 0.81 24 0.91 32 0.88 42 0.92 55 ' 0.91 102 0.54 103 0.71 126 0.94 127 1.08 128 0.91 132 1.08 Cont 1,00 Table Vd,. V i a b i l i t y index, growth at 27 C standardized against controls (set to 1.00). The index f o r the l i n e s was calculated as a mean of f i s s i o n rates over 10 days; the wild type controls averaged 4.43 f i s s i o n s per day. 25 I I E f f i c i e n c y of the Selection System (Marked-Recapture Experiment) The screening system was calibrated by marking a known t s mutant ( l i n e #2A2, iso l a t e d through the screen) with the behavioural gene pawn.^ Selections were carried out st a r t i n g with* 1, 10, or 100 pawn ts double 4 5 , 5 mutants i n 10 , 10 , and to x 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 to 1.2 x 10 , the mean over seven runs being 7»6 x 10J (Table V l ) . Thus, under average conditions with l i t t l e dessication, absence of crowding, and good quality l a b e l l e d bacteria f o r food, the screen enriched f o r t s mutants of the kind desired, 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 Recovi Pawn • 1 100pw/l.2xl0^/T pw/WT.=8.34xl0"3 25pw/NIL WT pw/WT.*25.0 >3.0xl03 25% 2 100pw/l.2xl0**WT pw/WT.=8.34x10-3 I6pw/NIL WT pw/WT.*16.0 > 1.9x103 . Wo 3 . 100pw/l.2xl05wT pw/WT.=8.34x10"^ 45pw/l6 WT pw/WT.=2.82 , 3«4xl02 Wo 4. 100pw/6.0xl05wT pw/WT.=1.67x10"^ 8pw/7 WT pw/WT.=l.l4 6.8x103 8% 5 10pw/l .2xl0 Z % pw / w r . - s^xio-^ 6pw/NIL WT pw/WT.*6.0 > 7.2x103 60% 6 lpvr/l . 2 x l o V r pw/WT.=8.34xl0"5 2pw/3 WT pw/WT.=0,67 8.0x103 200% 7 ipw/i . 2 x i o V r pw/WT.=8.34xl0~5 IPW/NIL vrr pw/WT."1.0 >1.2x10^ 100% Table VI. Range over 7 runs was 3.38xl0 2 to 1.2xlOLK The mean being 7.6lxl03 o pw=pawn marked ts2A2 c e l l s , WT=wild type c e l l s . 26 III Pattern of Decrease in Survival and Clone Forming; Ability  Due to Selection Just as temperature-sensitive periods usually precede death in most developmental lethal ts mutations, for example, in Drosophila melanogaster (Suzuki, 1970), so i t was expected that a dose of UV light large enough to destroy the cells' ability to form clones would be passed long before the cells actually diedo To test this, cells were subjected to varying exposures of light in otherwise standard selection runs. Cell density in the cultures was determined, and individual isolations were made. The vegetative lethality and clone forming ability 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 ability 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 far more heavily damaged than was obvious during the selection treatments. On the basis of these observations, light treatment was restricted to 12 hours. 27 FIGURE 4. Vegetative survival and clone formation  with varying UV light exposure. FIGURE 4 A (vegetative survival of -200- BU substituted cells) HOURS LIGHT EXPOSURE 28 IV Relative Contribution of Components of the Selection System  to Total Cell Death Because two critical periods in the selection procedure had been discovered (the threshold of cloning ability 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 cell fate. Firstly, experiment 6a (Table IV) was studied in detail because i t resulted in 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 light on the amount of exautogamous death and its role in 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 cell shapes other than normal were noted during the light exposure: the f i r s t was the "rounded-up" cell appearance that was known to precede lysis; the second was slightly pear shaped and vacuolated. The pear shaped cells were expected to die much later. 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 in death was realized in a l l groups. The viability of the exautogamous survivors was then checked. Wild type survivors grew well (all viables); however in the "dark" controls, seven percent of the survivors f e l l into the subvlable group. The experimentals were even worse than expectedi 17.2 percent of them were subviable. If the number of subviable lines is summed with the exautogamous deaths, i t is possible to see the total effect of the components of the screen. The wild type exautogamous death and subviability (11.8$) was increased to 35»0 percent by BU, and to 58.6 percent when irradiated. The influence of BU on micronuclei as distinguished from 29 that of UV light can also be seen at a l l points in the selection (Table Vllb). Both influences (BU, UV light) decrease the viability of exautogamous survivors by 23.2 and 23*6 percent respectively; these cells were either killed or rendered subviable. It appeared that the effect of both BU and of light was to preferentially k i l l rather than disable. The possibility that light alone might have a detrimental result on the cells was tested; for no "light-alone" controls were used i n the previous experiment. TABLE VII RELATIVE CONTRIBUTION OF COMPONENTS OF THE SELECTION SYSTEM TO TOTAL CELL DEATH Group Vegetative Death Exautog, Death Exautogamous Viability Survivor Viability* . Sub-viable Viable Exautog. Death+ Subviable Wild type (no treatment) Dark cont. (BU, no UV light) Experimental (BU, UV light) B Actual Dark- BU Effect (Dark minus wild type) Actual UV  Light Effect (Exptl. minus Dark) Table VII-A, breakdown of cell fate in the Selection System. *, clones were scored on a ranking scale - see Materials and Methods. Exautogamous death, and.Survivor viability categories add up to 100$. 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. 2.9$ 11.8$ 0 88.2$ 11.8$ 5 M 28.0$ 7.0$ 65.0$ 35.0$ 17,0% 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$ 30 The influence of light alone was determined by irradiating wild type cells in 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 ability. Samples of the 13 hour exposed cells (both intensities) were delayed in 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 in the DNA (on alkaline sucrose gradients) following irradiation (Humphrey et al. , 1970; Smets and Cornelius, 1971) as does Escherichia coli 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 ability was retained. In the experiment, no significant loss occurred in the samples exposed for 13 hours (Figure 5)« It 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 50 HOURS OF DELAY BEFORE ISOLATION 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. 3 2 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 in 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 in 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 hit damage rather than a single point mutation. If this is 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 in Sonnebom's methods paper (1970) but because i t is 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 in Materials and Methods). Indeed, mass cultures often may have doubled in 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 in the S phase of the cell cycle. There is no macronuclear G^  in 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 is highest in the S period of Escherichia coli (Cerda-Olmedo, Hanawalt, 33 and Guerola, 1968), in fact i t halts DNA synthesis immediately in that cell (Guerola, Ingraham, and Gerda-Olmedo, 1971). Cells will presumably be affected by NG no matter what their stage in the cell 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 coli (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 its inhibitory effect. However, since only about 18 percent of the population would be expected to be in micronuclear S period during exposure to the 11 mutagen, this effect would be relatively small. Mutation in macronuclear DNA was perhaps more common than in micronuclear since the S period is five times as long (occupying approximately 0.70 of the cell cycle). This implies 12 that almost 61 percent of the cells were treated in 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 cell line would in a l l probability have become a "slow grower" until the subsequent autogamy when the cells would probably have died due to micronuclear damage. After vegetative death few cells were lost until the completion of phenotypic lag. after autogamy. Sonneborn (1965) discussed the concept of "silent mutations"; those mutations which change DNA codons in 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 in Salmonella typhinurium the HisC gene of actually resulted in 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 cell to mutagenic damage. There was probably a large number of copies of each mutation in the cultures that were selected because homozygosed cells had to be grown for eight to 10 cell cycles after autogamy before expression of mutant phenotypes occurred (phenotypic lag). This duplication of mutants may have been beneficial i 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 in 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 in initiation of routine 35 crosses to be performed later. The f i r s t crosses should use lines between, not within batches. The number of sibs present in 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. If this period allowed 10 fissions, i t follows that there was up to 2^ copies (sibs) of a single mutational event present in the culture. In reality, this is unlikely because wild type cells will 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 in 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 in an increase in survivors, but a decrease in the number of putative ts 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 vital organelles. The effect of NG on mitochondrial DNA is unknown, as is 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 in Paramecium aurelia (Beale, .1969; Adoutte and Beisson, 1970, 1971; and Beale, Knowles, and Tait, 1972); and in 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 lines survive the treatment. I t i s possible that most mitochondria were regularly destroyed but sufficient numbers remained functional to allow the ce 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 cells that survived grew well immediately after l i g h t treatment. Thus this factor could not have been overwhelming. One of the advantages of the experimental design was that survivors of an inefficient selection (due to poorly labelled bacterial food perhaps) could be grown slightly to clear the cel l ' s metabolic pools of BU, and then retreated with BU substituted bacteria and irradiated again, to enrich for putative ts mutants. (Generally, a successful run l e f t so few survivors that reselection was totally unnecessary.) Whether reselection made the overall system more efficient 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 lines 14 i n isolates was significantly higher i n the reselected runs. The possibility 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 bacterial 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 lines, 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. Explanations for the existence of the category are difficult. In some cases autoradiographic data demonstrated an "on-off" characterT a sample of cells from a particular line contained individuals K i t h either very low incorporation ("off"), or a higher level of incorporation ("on"). The inference is that cells of such a line may be exhibiting less penetrance, or that the particular mutation confers a critical ts period on the cells (that were unsynchronized), for example, initiation. If the cell i s already in 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 in Paramecium will prove interesting. The Selection System only demands that the ts cell stop synthesizing DNA within one cell cyclei however, mutations that act much faster may be present. Other studies concerning related functions of the cell 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 in 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 cell generations to produce a cell with perhaps 70 percent of the normal DNA content before the cell reacts to the problem. This would not be easyj i t would be necessary to shift a series of synchronized cells to 38 the restrictive temperature just before termination of DNA synthesis. At each successive c e l l generation a set of synchronized cells would have to go through the same treatment. Representatives from each generation would be Feulgen stained and the relative amount of macronuclear DNA measured by microdensitometry (macronuclear DNA content would be estimated relative 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 deficient 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 in exconjugants. In addition, the mechanism by which fragment DNA synthesis i s normally selectively inhibited (Berger, 1973) following conjugation or autogamy could be examined. A ts DNA 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, ts/ts. A temperature s h i f t up to the restrictive level after conjugation may force the fragments of the old macronucleus (a r e l i c of the preconjugation macronu-cleus) to synthesize DNA, and form a substitute nucleus. The activation of the old fragments Would depend on a fast inhibition 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 for c e l l cycle studies. The usefulness of synchronized cultures has been well established. Hotchkiss (1954) developed a method for synchronizing the growth of bacteria, 39 and Scherbaum and Zeuthen (1954), using heat shock achieved 85 percent synchrony in Tetrahymena. These procedures opened new avenues to the study of DNA metabolism in Escherichia coli (Schbach and Davern, 1970; Rodriguez, Dalbey and Davern, 1973) and to the study of metabolic control and oral morphogenesis in Tetrahymena (Frankel, 1967)» Most recently, Hartwell and Hereford (1973) used ts mutations to synchronize yeast cells although somewhat unsuccessfully. No such breakthroughs have occurred in Paramecium. 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 in the cell cycle. Exautogamous or exponential cultures could be heat shocked for six to 12 hours (the time spent at the restrictive temperature in 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 in the collection of dividers. Concluding Statement In summary, cell lines exhibiting a l l levels of depression in DNA synthesis have been isolated utilizing 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 in eukaryotic organisms have yielded poorer results using replica-platingt in 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 ts tests prior to incorporation studies. 4 0 NOTES 1. "Ts mutant" i s used even though no g e n e t i c a n a l y s i s was performed because a l l t e s t e d c e l l l i n e s have been subsequent ly conf i rmed as gene c o n t r o l l e d ( r e c e s s i v e ) mutants . 2. Overn ight 10ml. c u l t u r e s were supplemented w i t h JO/jg/ml* o f thymine . Only lQtfg/ml. was used 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 washing) 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 o f 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 found t o be p o t e n t f o r use i n the s c r e e n i n g procedure 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 t o r e f r i g e r a t i o n d e s t r o y e d t h e 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 to s u f f i c i e n t medium. (Donachie and Hobbs, 1967). 5. The d e f i n i t i v e p roo f t h a t BU e n t e r s the macronucleus (and m i c r o -n u c l e u s ) might 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 ) which i f debrominated , would not l a b e l DNA. The BU must be i n f l u e n c e d by b a c t e r i a l polymerase and n u c l e a s e s and polymerases i n the paramec ia - i t i s p o s s i b l e t h a t the BU i s debrominated 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 (deoxyur id ine i s a ve ry e f f i c i e n t DNA p r e c u r s o r because i t can be conver ted to t h y m i d y l i c a c i d by t h y m i d y l a t e s y n t h e t a s e ) . B , t h a t BU g e t s i n t o the c i l i a t e DNA by the d e s c r i b e d t e c h n i q u e s , might be shown by s p i n n i n g down DNA f rom the 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 to demonstrate t h a t the heavy bromine 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 to ensure s a f e h a n d l i n g o f the mutagen. T h i s box was used w i t h i n a fumehood. 7. G e n e r a l l y , the t r e a t m e n t s o l u t i o n s were topped-up c a r e f u l l y w i t h d i s t i l l e d wate r . 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 the l e v e l was r e p l e n i s h e d too q u i c k l y . One attempt was made to m a i n t a i n the 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 reduces 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 the 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 the d e s i c c a t i o n when l e f t unat tended f o r over 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 cons tan t f l u i d depth apparatus was d e v i s e d based on the s i p h o n , however i t was no t w i d e l y implemented because o f u n r e l i a b i l i t y . 8. I f the t h r e s h o l d was not passed 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 , the c e l l s t i l l had an enormous o b s t a c l e ahead : autogamy w i l l be the Nemesis o f many i n d i v i d u a l s . ( Th is argument o f course h o l d s f o r m i t o c h o n d r i a , except t h a t the t h r e s h o l d f o r each o r g a n e l l e must be ext remely low - bu t the number o f o r g a n e l l e s i s h i g h . ) 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 . Exconjugates o f t h i s c r o s s were used to p i c k up F£ d i h y b r i d progeny. Ts 2A2, i s o l a t e d by the s e l e c t i o n sys tem, i n c o r p o r a t e s l i t t l e o r no 41 DNA precursor at 35°C. This t s also stops growth a f t e r about f i v e hours at the r e s t r i c t i v e temperature (Eric Peterson, personal communication). 10. Since the vegetative macronucleus i s highly p o l p l o i d , the c e l l could cope with serious genetic 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 exponentially growing asynchronous culture, the proportion of c e l l s that are i n micronuclear DNA synthesis can be approximated by use of the equation tm/T = M/0.693 or M X 1.44 (these equations have been used by Hoffman, 1949; Stanners and T i l l , I960; Smith and Dendy, 1962; and Painter and Marr, 1968| and are based on the curve y = 20-~x) where M = m i t o t i c index, t = c e l l cycle time, and tm «* the time i n mitosis a t the end of the c e l l cycle (tm can be used i f M i s small). Therefore, M = 35/300 minutes = 12 percent, tm/T = 0.12/0.693 = 0.12 x 1.44 = 0.173. According to these calculations presumably 17 percent of the c e l l s would be i n micronuclear S at any moment. This i s only an approximation since S f o r Paramecium micronuclei i s not a t the end df the c e l l cycle (there i s a subsequent 115 minute G^). A more accurate estimate can be calculated using the plot of y = 23-~x) which gives r e l a t i v e numbers of c e l l s at points i n the c e l l cycle (by calculating the area that micronuclear S occupies under the curve). This gives a number of 18 percent f o r micronuclear DNA synthesis. I f many c e l l s entered S i n the presence of the mutagen, the figure representing the portion of c e l l s treated i n micronuclear S i s conservative. 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 gives 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 ~ x ) ) gives 61 percent - a more r e a l i s t i c f i g u r e . This discrepancy proves the inadequacy of the f i r s t estimate when the duration of S i s too great. 13. Byrne (1973) has i s o l a t e d mutations which, when homozygous, produce s e l f i n g . Butzel (1953) f i r s t proposed that mating type substances (causing the mating reaction) 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 responsible f o r making an animal mating type VII; and E, mating type V I I I . Taub (1966) agreed with these findings. Byrne suggested that 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 blocks along the pathway to E substance. His findings are consistant with the observation that 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 or mating type VII to mating type V I I I . As an aside, a system of deliberate mating, following mutagenesis instead of autogamy, could be attempted to screen f o r dominants. 14. 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New Class of Purine Mutants of Chinese Hamster cells. Science. 172: 162-163. 48 Wacker, A., Dellweg, H., and Weinblum, D. ( l96l). Uber die 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. Whitfield, H., Martin, R., and B. Ames. (1966). C l a s s i f i c a t i o n of Amino transferase (C Gene) Mutants i n the Hi s t i d i n e Operon. J. Mol. B i o l . 211 335-355-Woodward, J . , Belber, B., and H. Swift. ( l 9 6 l ) . Nucleoprotein changes during the mitotic cycle 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" Escherichia c o l i @ 37° C In the growth curves, A was supplemented with Bacto-casitone, while B and C had Casamino acids. (2.5mg/ml.) which might lack some metabolite which was present i n casitone; thus the increased lag would have occurred while there was induction of biosynthetic pathways to make the product. The cultures were also supplemented with thymine (30 g/ml.), u r a c i l (15 g/ml.) and dextrose (5mg/ml.). In the selections, 24 g/ml. of 5-bromouracil was substituted f o r thymine. In a 10ml. culture, of culture, the proportions were 9ml. stock Pierucci's modified M-9 s a l t s , 50mg. dextrose, 25mg. casamino acids, 150 g. u r a c i l , and J00 g. thymine or 240 g. of 5-bromouracil. 15TAU~ was used i n selections 4b, 5a, 5b, 6a, and 6b (Table IV). The s t r a i n was also 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 putative t s l i n e s (except f o r #63 and #125) i n Table Va, part 1 of the Results. Growth Curves f o r 15T~ Escherichia c o l i @ 37°C A, B, and C were three separate cultures a l l exhibiting lengthy lag (245-3?5 minutes) before growth. The cultures were supplemented as with 15TAU". . 15T" was used i n selections 7a, 7b, 7c, 7d, and 7e (Table IV) as well as i n the vegetative dosage and clone forming a b i l i t y experiments. Section I I I of the Results. Growth Curves f o r 15T~55-7 Escherichia 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 ag i t a t i o n during these experiments. This 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 at 4°C, and ea s i l y r e i s o l a t e d from contaminated cultures. 15T~55"7 was used i n selections 10a, 10b, 10c, lOd, and lOe (Table IV), as we l l as i n the Paramecium ^H-BU uptake experiment (Table I I of the Materials and Methods), the Marked-Recapture experiment (Section I I of the Results), and the 3H-thymine test of DNA incorporation on putative t s l i n e s (Table Va, parts 2 and 3 of the Results). 50 APPENDIX I (growth curves) / 0.5-51 APPENDIX i: (growth curves) OA- / X S A / / B J C / o o.3- . E.coli 15T / x h S J / J J I J "^0.2 • « 5 J05 565 425 455 545 605 ' 665 ' 725 MINUTES Q5-°-4' thymine ^x' Ecoli 15T~55-7 X* n^°~°^° . ^^-°^°"~cr" 5-bromouracil. x-—x^ 0 ' 6 0 120 MINUTES 180 240 52 APPENDIX II Theoretical Scheme f o r the Use of t s DNA Mutants i n an Experiment Concerning the Control of DNA Metabolism The i n i t i a l cross of mutant by wil d type simply yie l d s heterozygotes which would be backcrossed to the mutant, giving an F£ with two classes of c e l l s i n each mating type. One class of c e l l s (cytoplasmically derived from the FT heterozygote) would have the genotype t s / t s , t s / t s , and ts/+ f o r the macronuclei, micronuclei, and macronuclear fragments respectively. 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, nuclei could not synthesize DNA. This s i t u a t i o n could be remedied i f the macronuclear fragments (genotype ts/+) "switched on" synthesis of DNA. This may force the c e l l to go through a forced macronuclear regeneration -a phenomenon only r a r e l y seen i n the l i f e cycle. 


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