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The isolation and phenotypic characterization of Paramecium tetraurelia temperature-sensitive cell cycle… Chua, Gordon 1994

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T H E I S O L A T I O N A N D P H E N O T Y P I C C H A R A C T E R I Z A T I O N O F PARAMECIUM TETRAURELIA T E M P E R A T U R E - S E N S I T I V E C E L L C Y C L E M U T A N T S G O R D O N C H U A B . S c , University of British Columbia, 1991 A THESIS S U B M I T T E D IN P A R T I A L F U L F I L L M E N T O F T H E R E Q U I R E M E N T S F O R T H E D E G R E E O F M A S T E R O F S C I E N C E in T H E F A C U L T Y O F G R A D U A T E STUDIES (Department of Zoology) We accept this thesis as conforming to the required standard T H E U N I V E R S I T Y O F BRITISH C O L U M B I A December, 1994 © Gordon Chua, 1994 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for schoiariy purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of TP^LCGJ The University of British Columbia Vancouver, Canada Date .95/01/13 DE-6 (2/88) ii Abstract. Conditional cell cycle mutants which block cell division under restrictive conditions, increase their cell mass as a result of continued growth. Here, we develop a protocol to screen for large Paramecium temperature-sensitive cell cycle mutants by size-selecting centrifugal elutriation. By using the ccl mutant as a standard for Paramecium temperature-sensitive cell cycle mutants, we determine that 14 hours of heat treatment produces sufficient size difference for the elutriator to discriminate cell cycle mutants from wild-type cells. In addition, this duration of heat treatment does not reduce the viability nor delays recovery rates significantly in cell cycle mutants. We demonstrate that multiple elutriations are able to retrieve ccl cells at cell cycle mutant frequencies of 0.02%. Two mutants, cc5 and cc8 have been isolated from this screen. Temperature-shift experiments reveal that these two mutants possess similar temperature-sensitive periods to ccl. The cc5 and cc8 mutants are allelic to each other but extragenic to ccl. The presence of a "pseudo" class in the F2 progeny of the cc5 X ccl cross suggests an interaction between the cc5 and ccl gene products. iii Table of Contents. Abstract ii Table of Contents iii List of Tables v List of Figures vi Acknowledgement vii I N T R O D U C T I O N 1 M A T E R I A L A N D M E T H O D S 8 1. Stocks and cultures 8 2. Recovery rates 8 3. Elutriations 9 4. Mutagenesis 12 5. Temperature shifts 13 6. Crosses 15 R E S U L T S 17 1. Effects of heat treatments on recovery rates of wild-type and ccl cells 17 2. Segregated elutriations of wild-type and ccl cells 19 3. Extended elutriations of wild-type and ccl cells 19 4. Population density effects on elutriation 21 5. Multiple elutriations of wild-type-cc7 mixed cultures 21 iv 6. Isolation of cell cycle mutants from mutagenesis 26 7. Transition points of ccl, cc5, and cc8 34 8. Temperature down shifts of ccl, cc5, and cc8 34 9. Matings between ccl, cc5, and cc8 42 D I S C U S S I O N 1. Enrichment of the cell cycle mutant screen 45 2. Reasons for low yield of ccl cells from wild-type-cc/ mixed cultures 45 3. Isolation of cell cycle mutants 50 4. Temperature-sensitive periods of ccl, cc5, and cc8 53 5. Genie relations between ccl, cc5, and cc8 55 6. Nature of "pseudo" cells 55 B I B L I O G R A P H Y 58 V List of Tables. Table 1 Retrieval of ccl cells from mixed wild-type and ccl cultures 25 Table 2 Effect of mutagen dose on induction of autogamy 27 Table 3 Percentage of lethality caused by mutagen dose 28 Table 4 Cell types retrieved from elutriations of mutagenized culture 30 Table 5 Phenotypic expression of cell cycle mutants at permissive and restrictive temperature 33 Table 6 Transition point determination by temperature down shifts 36 Table 7 Phenotypic ratios of F l and F2 progeny from genetic crosses 43 List of Figures. Figure 1 The effect of heat treatment duration on wild-type and ccl cells 18 Figure 2 Segregated elutriations of wild-type and ccl cells 20 Figure 3 Extended elutriations of wild-type and ccl cells 22 Figure 4 Population size effect on flow rate required to elute 50% of cells 23 Figure 5 Transition point of ccl 35 Figure 6 Temperature down shift and one hour heat pulse of wild-type cells 38 Figure 7 Temperature down shift of ccl 39 Figure 8 Temperature down shift of cc mutants 41 vii Acknowledgements. I sincerely thank Dr. James Berger for his valuable advice and continuous support during my Masters. In addition, I am grateful for his ability to see the potential in me in the beginning and allowing the development of it. I am also appreciative of the help received by my committee members Dr. H . Brock, Dr. T. Grigliatti, and Dr. D . Moerman as well as these people in the lab: Mike Adl, Liren Tang, Colin Ross, Mary Anne Muir, and Mike Wang . Finally, I would like to thank my family and my dear friend Carolyn Lee for their encouragement throughout my studies. 1 In order for a eukaryote cell to divide, three major tasks must be completed: (1) approximate doubling of cell mass; (2) replication of. chromatin and; (3) accurate distribution of cellular components to the progeny. The sequence of events by which this is accomplished is called the cell cycle. The eukaryote cell cycle is divided into four main phases: G l , S, G2, and M phases (preDNA synthesis, D N A synthesis, post-DNA synthesis, and mitotic phases respectively). During these phases, continuous processes such as mass accumulation and periodic events involved in replication and segregation of D N A are occurring. These two processes, known as the growth cycle and the D N A division sequence respectively, (Mitchison, 1971) are coordinated to accomplish proper regulation of cell mass and gene dosage, thus ensuring that each daughter cell possesses the appropriate size and D N A content. Coordination of cell growth and division occurs at two control points within the cell cycle: one in late G l called S T A R T and one at the onset of mitosis or the G 2 / M boundary (Nurse, 1975; Thuriaux et al., 1978). This is evident because progression of the cell cycle past these control points is dependent upon attaining a critical size (Johnston et al., 1977). S T A R T is a key decision-making point in the cell cycle at which cells either commit to a mitotic cycle or undergo differentiation leading to meiosis or mating (Nurse, 1981; Pringle and Hartwell, 1981), while the G 2 / M control point commits cells into mitosis and cell division after D N A replication (Nurse and Fantes, 1981). Studies that have identified key cell cycle regulators and their substrates involved in these two control points have enhanced our understanding in important biological areas such as cell proliferation, control of developmental choice, cellular senescence, apoptosis, and oncogenesis. A powerful approach to identify cell cycle regulators and their substrates is by isolation and analysis of conditional cell cycle mutants. Cell cycle mutants first isolated in Saccharomyces cerevisiae (Hartwell, 1971) are predominantly heat sensitive mutants. At the low or permissive temperature, the cells grow normally while at some higher, restrictive, temperature, they fail to undergo division. Although mutations in genes that 2 control growth and metabolism are capable of arresting cell cycle progression, the definition of a cell cycle mutation is restricted to those mutations that lead to a defect in a stage specific event (or landmark) that occurs once per cell cycle, such as D N A replication or nuclear division (Hartwell, 1978). Thus, an asynchronous cell cycle mutant population eventually becomes arrested at one common cell cycle landmark after incubation at the restrictive temperature for a time. Another common trait found in cell cycle mutants is their acquisition of an abnormally large cell mass after prolonged exposure to heat treatment. This is a consequence of uncoupling the growth cycle from the DNA-division cycle as the cells arrest at a landmark but are able to continue growth at the restrictive temperature (Johnston et al., 1977). Cell cycle mutants have been discovered in a variety of organisms other than Saccharomyces cerevisiae such as Schizosaccharomyces pombe (Nurse et al., 1976), Aspergillus nidulans (Orr and Rosenberger, 1976), Chlamydomonas (Howell and Naliboff, 1973), Tetrahymena (Frankel et al., 1976), Paramecium (Peterson and Berger, 1976), and mammalian cell lines (Thompson et al., 1970; Wang, 1974). One benefit offered by studying temperature-sensitive cell cycle mutants is the ease of determining the temporal location of the mutations in the division cycle. A series of temperature shifts can be performed simply on synchronous cell cycle mutant populations to resolve the time at which the mutant gene products exert their phenotypic effects (Frankel et al., 1980), rather than using drugs which are expensive and may produce secondary effects (Baserga, 1985), or the laborious task of isolating the gene. With temperature-sensitive mutants, the end of the temperature-sensitive period, termed the execution or transition point, is determined by shifts from permissive to restrictive temperature throughout the cell cycle (Hartwell, 1978). Cells that are exposed to restrictive conditions prior to the transition point are incapable of division, but after this point the succeeding division remains unaffected. The transition point is defined as the time in the cell cycle when 50% of the cells acquire the ability to divide following the shift to restrictive temperature (Frankel et al., 1980). In contrast, the beginning of the 3 temperature-sensitive period is identified by performing the reciprocal temperature shift, where cells are heat treated shortly after fission and returned to permissive conditions at various points in the cell cycle (Esposito et al., 1970). Cells transferred to the permissive temperature prior to the temperature-sensitive period remain unaffected in cell division, while a delay in cell division proportional to the duration of exposure to restrictive temperature occurs in cells down shifted after the beginning of the temperature-sensitive period. The ciliate Paramecium possesses several features favorable for cell cycle research. As a free-living unicellular eukaryote, Paramecium can be cultured in medium similar to its natural habitat, thus minimizing disruption to growth. In addition, these organisms are fairly large, which enables experiments with single isolated cells, and so effects arising from cell-cell interactions are eliminated. Two other properties essential in cell cycle studies that paramecia exhibit include a short cell cycle approximately six hours long, and a high degree of synchrony that is easily obtained with a low level of perturbation. Paramecia are also suitable for mutational studies because they are quite responsive to a number of mutagens, and some species such as Paramecium tetraurelia are able to undergo autogamy, a cytogenetic process induced by starvation that results in complete homozygosis of all loci (Sonneborn, 1974). In Paramecium, the cell cycle is complicated by the presence of two nuclei types, macronuclei and micronuclei, each having unique functions and D N A division cycles. Macronuclei are polygenomic and transcriptionally active, thus controlling the cell's phenotype. In contrast, micronuclei are diploid, transcriptionally silent, and function as germinal nuclei. They undergo meiosis and fertilization to form a zygotic nucleus called a synkaryon which eventually differentiates into a micronucleus and a macronucleus. Furthermore, micronuclei also participate in the somatic process of stomatogenesis (Ng and Tam, 1987). Macronuclear S phase occurs from 0.25 to 0.90 in the cell cycle, followed immediately by amitosis as there is no discrete macronuclear G2 period (Berger 4 and Kimball, 1964; Tucker et al., 1980). Micronuclear D N A replication extends from 0.47 to 0.69 in the cell cycle and micronuclei divide mitotically (Pasternak, 1967). Similar to the macronucleus, the micronucleus division cycle has no G2 interval (Pasternak, 1967; Adl and Berger, 1992). Much of our understanding of the cell cycle in Paramecium has been provided by analysis of the temperature-sensitive cell cycle mutant ccl (Peterson and Berger, 1976). ccl cells arrest in both G l and S phase in the vegetative cell cycle, but cell growth continues at the restrictive temperature (Rasmussen and Berger, 1982, 1984). As a result, ccl cells exposed to restrictive temperatures attain sizes more than three times larger than wild type cells. However, the ccl mutation displays various characteristics distinct from other cell cycle mutations (Rasmussen and Berger, 1984). Firstly, this mutation blocks cell cycle progression completely with no residual progression and no excess delay of subsequent cell cycle events caused from exposure to heat treatment. This is supported by the observation that ccl cells arrested in G l for various time periods, and then released from the cell cycle block by temperature down shift, delay D N A synthesis initiation by the duration of heat treatment and the remaining portion of G l from the start of heat treatment. Secondly, unlike other D N A synthesis mutants (Pringle and Hartwell, 1983; Mitchison, 1971), the ccl mutation blocks macronuclear D N A synthesis rapidly and completely within 20 minutes after shift to restrictive temperature, and D N A synthesis begins quickly with similar kinetics upon return to permissive conditions. Thirdly, besides blocking macronuclear D N A synthesis, Adl and Berger (1991, 1992) have shown that the ccl defect obstructs other cell cycle processes such as oral morphogenesis and micronuclear spindle elongation. However, these cell cycle processes which also occur in the sexual pathway (conjugation or autogamy) are unaffected in ccl mutants, suggesting a difference in vegetative and sexual cell cycle regulation in Paramecium (Adl and Berger, 1994). In addition, micronuclear D N A replication proceeds normally in ccl mutants 5 under restrictive conditions revealing that D N A synthesis regulation in macronuclei and micronuclei occurs dissimilarly (Adl and Berger, 1992). The point of commitment to cell division in Paramecium is resolved by determining the transition point for the ccl mutation (Berger, 1988). Temperature up shifts of synchronous ccl cell samples to restrictive temperature at various times in the cell cycle indicate that the transition point occurs at approximately 0.75 in the cell cycle (Peterson and Berger, 1976; Rasmussen et al., 1985). This control point in the Paramecium cell cycle which irreversibly commits cells to division is also responsible for other key regulatory decisions regarding the next cell cycle. The timing of macronuclear D N A synthesis initiation is established about 90 minutes prior to fission in well-fed cells coinciding with the point of commitment to division (Berger and Ching, 1988). However, the timing of macronuclear D N A synthesis initiation is set in the preceding cell cycle rather than the present cell cycle, as indicated by experiments that examine the effect of increased cell mass on the timing of macronuclear D N A synthesis initiation. Cell mass perturbations produced by heat treating ccl cells accelerate the initiation of macronuclear D N A synthesis only if the increase in cell mass occurred in the preceding cell cycle (Rasmussen et al., 1986). The initial point of commitment to meiosis (autogamy) is also tightly coupled to commitment to cell division, because cell cycle perturbation by ccl heat treatment delays the initial point of commitment to autogamy to the same degree as the point of commitment to cell division (Berger, 1986). Thus, like other eukaryotes, the Paramecium cell cycle control system also governs entry to D N A synthesis, alternative pathways of mating or meiosis, and commitment to mitosis. However, in contrast to other eukaryotes in which the cell cycle control system acts at two discrete points in the cell cycle at late G l and the G 2 / M transition, the Paramecium cell cycle control system operates only at one cell cycle point, the point of commitment to division (Berger, 1988). Since research using the ccl mutant has generated considerable information on Paramecium cell cycle control, isolation of new cell cycle mutants may provide further 6 insight in this area. In general, temperature-sensitive cell cycle mutants are selected on the basis of their arrest of division after incubation at restrictive temperature. A few methods have been developed to enrich for temperature-sensitive cell cycle mutants within a mutagenized cell population. Most of these methods involve shifting cells to restrictive conditions and exposing them to agents that kill dividing cells, while temperature-sensitive mutants being growth and division arrested, survive and grow out again when the cultures are shifted back to permissive conditions in the absence of these agents (Basilico, 1978). However, these techniques actually select for growth and thus, cell cycle mutants are not discriminated from other temperature-sensitive growth mutants. Since the common feature of temperature-sensitive cell cycle mutants is cell cycle arrest under restrictive conditions with a concurrent increase in cell size, an alternative to enrich for this mutant type is to use a method capable of selecting larger cells. This selection scheme produces better separation of temperature-sensitive cell cycle mutants from growth mutants because the latter being defective in metabolism and growth tend to remain the same size or become smaller upon prolonged exposure to restrictive conditions. One possible method of isolating larger cells from a cell culture is via centrifugal elutriation ("counterflow centrifugation"). This technique has proven successful in cell size selection of yeast (Gordon and Elliot, 1977), red blood cells (Fignor et al., 1983), Tetrahymena (Seyfert et al., 1984), and Paramecium (Tang and Berger, 1994 ; Adl and Berger, 1994). Although utilized mostly to obtain synchronous cell fractions from mass cultures, centrifugal elutriation has contributed to the isolation of the temperature-sensitive Tetrahymena cell size mutant D9 which exhibits an increase in cell size by a factor of 2.5 at restrictive temperature (Seyfert et al., 1984). Briefly, centrifugal elutriation involves two opposing forces: (1) a constant centrifugal force that pushes cells away from the center of rotation; and (2) a fluid force that pushes cells towards the center of rotation. Because the elutriation chamber is funnel-shaped and the fluid containing the cells enters from the narrow end, the fluid force decreases as the chamber walls become wider. The 7 resulting fluid force gradient from the narrow end to the widest part of the chamber causes a gradual size separation of cells, with smaller and larger cells accumulating at the chamber's wide and narrow ends respectively. By increasing the flow rate (fluid force) or decreasing the rotor speed (centrifugal force) in gradual steps, successive fractions of increasing large cells are washed out of the chamber and collected. The focus of my research is divided in two parts: (1) the development of a protocol to screen for Paramecium temperature-sensitive cell cycle mutants by size selecting centrifugal elutriation; and preliminary phenotypic characterization of possible cell cycle mutants recovered from the protocol. The ccl mutant was used as a standard for Paramecium temperature-sensitive cell cycle mutants to test for the effectiveness of cell cycle enrichment by the proposed screening method. Initially, the optimum duration of exposure to restrictive temperature was examined using ccl cells. This was followed by separate elutriations of heat-treated wild-type and ccl cells to determine whether centrifugal elutriation was able to discriminate between the two cell types. Conditions that might influence the resolution of cell size separation, such as cell load and flow rate increments were analyzed and led to refinement of the method. A n elutriation trial run on a mixed cell population consisting of wild-type and ccl cells present at cell cycle mutant frequencies was performed before the actual mutagenesis to assess the screen's ability to retrieve cell cycle mutants from a cell population. Seven cell lines designated cc3-cc9 exhibiting temperature-sensitive cell cycle mutant characteristics such as failure to divide and increases in cell mass when exposed to restrictive temperature were recovered from a mutagenized cell population. The majority of these mutants possessed aberrant phenotypes at permissive temperature, including retarded growth, and they eventually perished. However, two mutant cell lines, cc5 and cc8, showed normal growth during permissive conditions, and thus, were analyzed further. Preliminary characterization of cc5 and cc8 involved defining the temporal position of their temperature-sensitive period in the cell cycle and the genetic relationship between each other and ccl. 8 Material and Methods. Stocks and culture conditions. Stock cultures used for these experiments were Paramecium tetraurelia 5 IS (wild type, Sonneborn, 1975) ccl (line 2A2, Peterson and Berger, 1976) and paranoic (Kung, 1971). Paramecia were grown in phosphate-buffered Cerophyl medium (Sonneborn, 1970) suppiemented with 0.5 mg/L of stigmasterol (Sigma) and innoculated the day before use with Klebsiella pneumoniae, the food organism. Cells were maintained as daily isolation lines in depression slide wells and grown at 27°C unless otherwise indicated. The cell cycle mutant phenotype was detected by placement of cells in either an air incubator or a water bath set at restrictive temperature (33.8-34.0°C). The air incubator, though less precisely regulated than the water bath, was used to screen large cell numbers because cells could be transferred more rapidly into depression slide wells than 13 x 100 mm glass test tubes. However, the water bath was used for more sensitive experiments such as temperature shifts to determine the temperature-sensitive periods and phenotypic analysis of mating crosses. Determination of ccl recovery rates. The ccl recovery rate was defined as the number of cell fissions occurring in a 24 hour period at permissive conditions (27°C) following exposure to restrictive temperature (34.0°C). To determine the maximum heat treatment duration that ccl cells could tolerate without compromising their recovery rates, ccl cells were exposed to heat treatment for various times and their recovery rates examined. Synchronous cell samples of wild type and ccl were obtained by manual selection of dividing cells with a micropipette. Paramecia were grown in 100 x 15 mm plastic Petri dishes containing 30-35 ml of Cerophyl medium overnight, and they reached cell densities of 800-1000 cells by the following morning. Each synchronous sample contained 80 newly divided cells which were 9 separated into two groups: an experimental and control group. The cells from the experimental group were transferred manually with a micropipette into a 13 x 100 mm glass test tube containing 2 ml of Cerophyl medium and submerged to a depth of 4 cm in a water bath set at 3 4 . 0 ° C for 6-24 hours. The water bath was capable of maintaining the desired temperature within 0 . 0 2 ° C . After heat treatment, the cells were removed from the test tube by a Pasteur pipette, and 30 cells selected at random with a micropipette were placed in a depression slide well with fresh Cerophyl medium. These cells were then stored at permissive conditions in a 2 7 ° C air incubator for 24 hours. After this time period, the number of cell divisions that occurred in each cell line was recorded. The control group was kept at 2 7 ° C throughout the experiment and handled in the same manner as the experimental group. The effect of heat treatment duration on recovery rates of wild-type and ccl cells were compared and graphed. Cell size separation by centrifugal elutriation. Initially, elutriation trials were performed on heat-treated wild-type and ccl cells separately to determine whether their size differences could be discriminated. Both wild-type and ccl cultures were grown from postautogamous cells in isolation lines under well-fed conditions where cell densities did not exceed 1500 cells/ml. Cell densities were estimated by manually counting the cells. Samples of 0.01 to 0.2 ml were removed with an Eppendorf pipetter from a thoroughly stirred culture. The cells were ejected into 1 ml of Cerophyl medium in a depression slide well and counted manually by withdrawing the cells one by one with a micropipette. Each count was the mean of three samples. As the cell population increased, the cell culture was transferred each day into progressively larger Erlenmeyer flasks containing more medium. When approximately 1.0xl0 6 -1.5xl0 6 cells had accumulated in a 2800 ml wide bottom culture flask containing 2000 ml of Cerophyl medium, they were centrifuged in 120 ml oil-testing tubes at 1800 rpm for 25 seconds and resuspended in 500 ml of fresh medium. The cell densities were adjusted to 300 and 800 10 cells/ml for wild-type and ccl cells respectively because these densities maintained the cells under well-fed conditions after 14 hours of heat treatment. 1500 ml of each cell culture type was transferred into 1000 ml flasks in 500 ml aliquots and incubated in the 3 4 . 0 ° C water bath for 14 hours. After heat treatment, the cells were centrifuged and concentrated in 300 ml of fresh Cerophyl medium. Between 6.5xl0 5 and 1.4xl0 6 cells were loaded by a Masterflex 745 pump (Cole-Parmer) at a flow rate of 38 ml/min into the small 4 ml chamber of a JE 5.0 elutriator rotor (Beckman) driven by a Beckman centrifuge (J6 series) spinning at 900 rpm. After all the cells were loaded into the chamber, fresh Cerophyl medium was continuously pumped in for the duration of the experiment. The flow rate was slowly increased to 45 ml/min where fractions were initially collected in 18 x 150 mm glass culture tubes as the cells began to elute out of the chamber. The flow rate was increased in 2 ml/min increments after every tube (25 ml) of eluted cells were collected until 103 ml/min flow rate where all the cells were eluted out of the chamber. After the elutriation process was completed, the cell density was estimated in the 25 ml aliquots of cell culture gathered at each flow rate by manual count as previously described. Then, the cells/ml, cells/aliquot, cumulative number of cells, and finally the cumulative percentage of cells/aliquot were calculated and graphed to obtained elutriation profiles of wild type and ccl. These elutriation profiles were compared to determine whether both cell types eluted from the chamber at different flow rates. Extended elutriations were performed to improve wild-type and ccl separation and to remove more wild-type cells from the chamber at low flow rates. Wild-type and ccl cultures were grown, elutriated, and profiles determined as above. However, at flow rates at which wild-type cells eluted out while the majority of ccl cells remained in the chamber, several 25 ml aliquots were collected. At flow rates of 59, 61, 63, and 65 ml/min, 5, 7, 14, and 14 of 25 ml aliquots were gathered respectively. 11 Retrieval of ccl cells from wild-type-ccl mixed cultures. Wild-type and ccl cells were grown and heat-treated as before. However, prior to elutriation, varying numbers of heat-treated ccl cells were added to a heat-treated wild-type culture. Approximately l .OxlO 6 cells with wild-type to ccl ratios of 10:1, 100:1, and 5000:1 were subjected to multiple extended elutriations. After elutriation, the latter fractions (67-103 ml/min) containing the larger cells were poured into 100 x 15 mm plastic Petri dishes and examined under a dissecting microscope. Large cells that resembled the arrested ccl phenotype were selected by a micropipette and transferred singly into depression slide wells containing 1 ml of fresh Cerophyl medium. These isolates were stored for 2.5 days in the 2 7 ° C air incubator to allow recovery from heat treatment. Subsequently, 10 cells from each isolate were transferred by micropipette into a new depression slide well with 1 ml of fresh Cerophyl medium and tested for the ccl phenotype in a 3 4 . 0 ° C air incubator for 16-20 hours. Cell lines that failed to double their numbers during the heat treatment were designated ccl, while those that divided more than once were scored as wild type. Wild-type and ccl cells were tested simultaneously. Control wild-type and ccl cells were used to check the temperature's ability to cause cell cycle arrest in ccl cells. The remaining cells in the latter fractions from the first elutriation (about 5% of the culture) were resuspended in 1200 ml of fresh Cerophyl medium and placed in two 1000 ml flasks. These cells were left in the 17°C air incubator for 2 days to permit recovery and proliferation before the next heat treatment and elutriation. Large cell isolates were selected only from the first elutriation fractions of 10:1 and 100:1 wild-type to ccl cultures. The 5000:1 wild-type to ccl culture was subjected to five elutriations, but only the latter fractions from the third and fifth elutriations were used to retrieve ccl cells. 12 Mutagenesis and selection of cell cycle mutants. A wild-type culture was grown from a single postautogamous cell under well-fed conditions to a population size of approximately 1.2xl0 6 cells. This cell culture was concentrated by centrifugation, resuspended in 240 ml of Cerophyl medium and divided equally into two smaller cultures. Mutations were induced by treating these two cultures with N'-methyl-N-nitro-N-nitrosoguanidine (Sigma) at 1.0 ug/ml and 2.0 ug/ml for 6 hours with thorough stirring in a 2 7 ° C shaker. After 6 hours, each batch of mutagenized cells was washed thrice in Dryl solution by centrifugation, and then suspended in 125 ml of old Cerophyl medium. The two cultures were each divided into three independent subcultures to facilitate complementation analysis if cell cycle mutants were recovered. The cells were then permitted to starve for 4 days in the 2 7 ° C air incubator and to undergo autogamy to allow expression of recessive mutations present. After the majority of cells had entered autogamy as detected by Dippell stain (Sonneborn, 1970), 210 cell isolates were selected randomly from each mutagen-treated culture and an untreated control. These cells were then grown for 2.5 days in depression slide wells containing 1 ml of Cerophyl medium at 2 7 ° C to determine the proportion of lethality caused by the mutagen dosage as an indication of the effectiveness of the mutagen. The cell culture exposed to the mutagen dosage which produced 30-40% lethality in the cell isolates was chosen. Following selection of the cell culture with the appropriate mutagen dosage, one of the subcultures was grown and elutriated several times as mentioned previously, while the other two remaining fractions were stored in the 17°C air incubator for later screens. A n average of 250 large cells were selected from the latter cell fractions of each of the five elutriations performed on the mutagenized cell population. Large cells that exhibited abnormal appearances other than those seen in ccl were also selected. The isolates were grown and tested for their ability to divide in the 3 4 . 0 ° C air incubator as above. The nondividers at restrictive temperature were divided into 5 classes: (1) cells that died under restrictive conditions; (2) cells that were smaller than controls incubated at 13 permissive temperature; (3) cells similar in size to controls; (4) cells of normal size with an abnormal shape and; (5) cells that were larger than controls. Because the first four categories did not represent cell cycle mutants, they were not maintained and eventually discarded. However, the latter category was retained as putative mutants for further analysis because they exhibited some characteristics of cell cycle mutants. Cell cycle arrest of cc mutants at restrictive temperature. The putative cc mutants were tested repeatedly for their inability to divide accompanied by continued growth during heat treatment. Samples of 5 dividers raised in 100 x 15 mm plastic Petri dishes with Cerophyl medium and selected as before were placed in 13 x 100 mm glass test tubes with 2 ml of medium and incubated in the 3 4 . 0 ° C water bath for 16-18 hours. The cell numbers were counted manually after heat treatment and compared to their control counterparts stored in the 2 7 ° C air incubator for the same duration as the heat treatment. Wild-type and ccl cells were tested concurrently as positive and negative controls at both temperatures for the same period of time. Temperature shifts of cc mutants. Temperature shift-up experiments were used to determine the transition point of cc5 and cc8. Because these experiments required many dividers, a 100 x 15 mm plastic Petri dish containing 30-35 ml of Cerophyl medium was inoculated with well-fed postautogamous paramecia at a cell density of 400-500 cells/ml late at night. This ensured that the cells were in early log phase at a considerable cell density the next morning. Each synchronous cell sample consisted of 28 dividers with deep fission furrows that were manually selected during a 5 minute interval. The time at completion of each sample was recorded and taken as to the beginning of the cell cycle. To improve synchrony, 50 recently divided cells from each sample within 10 minutes after the time was recorded were transferred manually into 1.5 ml microcentrifuge tubes containing 0.5 ml of Cerophyl 14 medium preincubated at 2 7 ° C . The remaining cells in the sample, which included those that had not divided, were discarded. The experimental samples were stored in the 2 7 ° C air incubator and temperature up shifted at 1 hour intervals from the start of the cell cycle by submerging the microcentrifuge tubes completely in the 3 3 . 8 ° C water bath. The control sample consisted of newly divided cells transferred individually to a glass plate holding 42 depression slide wells each containing 3 drops of Cerophyl medium. The controls were kept at permissive temperature in the 2 7 ° C air incubator throughout the experiment to determine the cell cycle duration. After 4.5 hours into the cell cycle, the controls were periodically examined at 10-20 minute intervals under the dissecting microscope, and the number of divided cells was recorded. The process was continued until all controls had divided. After 12 hours, the experimental samples were removed from the 3 3 . 8 ° C water bath, their contents poured into separate depression slide wells, and their numbers counted manually. The transition point was then determined by defining the time in the cell cycle at which a shift from permissive to restrictive conditions permitted 50% of the cells to successfully complete their cell cycle and undergo division. Because the temperature up shifts were initially performed at 1 hour intervals, the location of the transition point was refined by reducing the temperature shift intervals to 20 minutes. The percentage of cells having reached division was plotted as a function of time for both control and experimental data. The transition point was expressed as a fraction of the control cell cycle duration which was defined as the median time of cell division. The ccl transition point was also determined to identify the commitment point to cell division (Peterson and Berger, 1976; Rasmussen and Berger, 1984), to make comparisons to cc5 and cc8 transition points, and to assay the reliability of the experimental techniques. The onset of the temperature-sensitive period was determined in temperature down-shift experiments. Synchronous cell samples were retrieved from the same Petri dish used to obtain dividers for the temperature up-shift experiments. Similarly, each synchronous sample consisted of 50 newly divided cells placed in a 1.5 ml microcentrifuge 15 tube with 0.5 ml of Cerophyl medium. However, the experimental samples were transferred into a 3 3 . 8 ° C water bath at 20 minutes into the cell cycle and incubated for 1, 2, 3, 4, and 5 hours. After heat treatment, the experimental samples were temperature down shifted by transplantation into depression slide wells and returned to 2 7 ° C . When the controls began to divide, the experimental samples were also inspected under the dissecting microscope for cell division. The period required for the median of cell division was specified as the experimental cell cycle duration. The effect of heat treatment length on the cc mutant cell cycle duration was determined by graph analysis as previously described by Rasmussen and Berger, 1982, and compared to temperature down-shift results of wild type and cc7. A double-shift from permissive to restrictive temperature and then back again was executed to further define the temperature-sensitive period. However, these experiments were confined to wild-type cells only for reasons explained later in the "Results" section. The procedures used here were very similar to those of the temperature down-shift experiments. The only difference was that the synchronous experimental samples were shifted to restrictive temperature ( 3 3 . 8 ° C ) and heat pulsed for an hour at every hour into the cell cycle until 5 hours after the start of the cell cycle. Genetic analysis of cc mutants. cc5 and cc8 were initially crossed to wild-type cells to determine: (1) whether the mutations were dominant or recessive and; (2) to acquire both mating types of each mutant which were used for later crosses. To ensure a high success in mating, young postautogamous parental cells were used. Second-day left over isolation line cultures of cc mutants and both mating types of wild-type cells carrying the paranoic marker were mixed in depression slide wells. The cells were allowed to starve in the 2 7 ° C incubator where they became mating reactive at 6-10 hours after mixing. Twenty-eight tight conjugating pairs were selected manually and each placed in depression slide wells containing 3 drops 16 of Cerophyl medium. They were placed in the 2 7 ° C incubator for 10 hours to allow mating completion and separation. The heterozygous F l exconjugants were maintained separately in daily isolation lines for 12 fissions to allow for phenotypic lag and then tested for temperature sensitivity. Thirty second-day left over cells were transferred into 13 x 100 mm glass test tubes containing 2 ml of Cerophyl medium and heat treated in the 3 4 ° C water bath for 18 hours accompanied by concurrent control testing of wild-type and cell cycle mutant lines. Temperature sensitivity was scored as previously mentioned. The F2 generation was produced by permitting the F l heterozygotes to undergo autogamy by starvation. These postautogamous cells were initially checked for cross-fertilization by the presence of the semidominant paranoic marker in both exconjugant clones. The paranoic marker was detected by the continuous backward swimming of paramecia for 5 seconds when the depression slide was gently shaken. Eighty-four F2 exautogamonts were selected randomly from F l exconjugants (42 from each exconjugant clone) that revealed cross-fertilization and grown in isolation lines in the 2 7 ° C air incubator. The F2 lines were tested for temperature sensitivity as above after 12 fissions. The presence of the paranoic marker was also determined in each F2 line to ensure occurrence of cross-fertilization and normal segregation of the marker. Mutant lines of each mating type were selected and carried as stocks. Complementation tests were next performed to determine if cc5 and cc8 were allelic to ccl and to each other. Crosses between all 3 cc mutants involved a paranoic cc parent and another cc mutant of complementary mating type. These crosses and the phenotypic testing of the progeny were carried out as described above. 17 Results. Effects of varying heat treatment durations on recovery rates. There are two conflicting factors that require optimization in selecting the duration of heat treatment of cell population prior to screening: (1) longer heat treatments allow for greater size difference between possible cell cycle mutants and normal cells and; (2) longer heat treatments reduce viability of the cultures and delay their recovery after return to normal culture temperature. Based on the assumption that Paramecium cell cycle mutants would behave similarly to ccl in response to heat treatments, ccl cells were used to determine the effect of heat treatments on recovery rates. Wild-type and ccl cells were heat treated for various times and their recovery rates measured by noting the number of cell divisions that occurred after down shift to permissive temperature. Untreated wild-type and ccl cells were used as standards to detect the presence of heat treatment duration effects on cell ability to recover. Increasing heat treatment duration retarded recovery rates of both wild-type and ccl cells, but the latter was affected more severely than wild type (Fig. 1). In ccl cells, recovery rates resembled those of untreated controls until 14 hours of heat treatment. After 14 hours, ccl recovery rates decreased dramatically, down to 25% after 24 hours of heat treatment. In contrast, heat-treated wild-type cells survived better than ccl cells, as their recovery rates were not affected until 18 hours of heat treatment and decreased gradually to 81% after 24 hours of heat treatment. The dramatic decrease in ccl recovery rates was probably caused by extensive damage as cells continued to grow without dividing. This was evident as ccl cells subjected to prolonged exposure to restrictive temperature appeared darker than normal and displayed aberrant shapes such as an enlarged posterior and occasionally a spherical appearance. The slight decrease in wild-type recovery rates that occurred after heat treatment durations greater than 18 hours was probably due to some heat treatment effects. Although their cell shapes appear normal, 18 4 9 14 19 24 Hours at restrictive temperature (34C) 19 these wild-type cells were slightly larger and darker than those cells not heat treated or heat treated for a shorter time. These results indicated that the maximal duration of heat treatment possible without compromising recovery in ccl cells was about 14 hours. Size discrimination of wild-type and ccl cells by centrifugal elutriation. Upon discovering the optimum heat treatment duration, segregated elutriation trials of heat-treated wild-type and ccl cells were performed to determine whether the increase in ccl cell size caused by 14 hours of heat treatment was sufficient to be discriminated by elutriation from wild-type cells. The difference in the elutriation kinetics of both cell types indicated that large heat-treated ccl cells could be separated from wild-type cells by elutriation (Fig. 2). Wild-type cells eluted from the elutriation chamber at lower flow rates than did ccl cells. The majority of wild-type cells (-98%) were removed from the chamber at the flow rate of 79 rnl/min, whereas the same proportion of ccl cells eluted out at 87 ml/min. The elutriation profiles in Fig. 2 also revealed the flow rates that produced the highest ccl to wild-type ratios. Cell fractions collected at flow rates greater than 75 ml/min contained 78% of ccl cells and 20% of wild-type cells. This data was informative because the cell fractions eluted at these specific flow rates were highly enriched with large cells and were used to select possible cell cycle mutants during the screen. Together, these results showed that a Paramecium cell cycle mutant screen using elutriation was possible. Improvements in ccl enrichment by extended elutriations. In the initial separate elutriation trials of wild-type and ccl cells, cells were collected by increasing the flow rate at 2 ml/min increments per 25 ml aliquot. However, not all the cells capable of eluting from the chamber at a specific flow rate were collected in a 25 ml aliquot. Therefore, extended elutriations were used whereby more than one 25 ml aliquot were gathered at a specific flow rate to allow collection of a larger fraction of 20 Fig. 2: Segregated Elutriations of Wild-Type & ccl Cells. 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 Flow rate (ml/min) 21 the cells capable of eluting at that flow rate. In an attempt to improve enrichment for ccl cells, extended elutriations on separate wild-type and ccl cell cultures were performed involving the collection of 5, 7, 14, and 14 25 ml aliquots at flow rates 59, 61, 63, and 65 ml/min, respectively. These flow rates resulted in elution of most of the wild-type cells, while ccl cells remained in the chamber. Although both wild-type and ccl cells were removed from the chamber at lower flow rates compared to the previous elutriation trials, extended elutriation results showed better separation. Cell fractions collected after 65ml/min contained 81% and 10% of ccl and wild-type cells originally present respectively (Fig. 3) Population density effects on elutriation. Examination of data from the previous elutriations showed that the cell density in the elutriation chamber affected the flow rates required to elute both wild-type and ccl cells. As the cell population in the chamber increased in elutriation runs of wild-type and ccl cells, higher flow rates were required to elute the same proportion of cells (Fig. 4). This was probably due to clumping of cells and poor cell size separation caused by the extreme tight packing of cells in the chamber. Thus, in subsequent elutriation experiments, population sizes of 1.0-1.2x106 cells were used to ensure the flow rates required to elute out a specific proportion of cells remained constant and to maintain adequate cell size separation. Isolation of ccl cells from wild-type-ccl mixed cultures by multiple elutriations. A further strategy to improve the mutant screen was the use of multiple elutriations on a mutagenized cell culture. This technique involved the collection of latter fractions which contained larger cells from the previous elutriations and subjecting them to further elutriations a few days later. Multiple elutriations were advantageous for two reasons. First, any large mutant cells that remained undiscovered after the first elutriation 22 Fig. 3: Extended Elutriations of of Wild-Type & c d Cells. 41 45 49 53 57 61 65 69 73 77 81 85 89 93 97 101 105 109 Flow rate (ml/min) Fig. 4: Population Size Effect on Flow Rate Required to Bute 50% of Cells. 80 i - 7 8 60 -I 0 200 400 600 800 1000 1200 1400 1600 Population size (1000's) 24 were allowed to multiply in numbers before the next elutriation. Therefore, the probability of isolating these mutants increased in subsequent elutriations. Second, certain mutants would recover quickly after they were removed from restrictive temperature. Recovered mutant cells were extremely difficult to distinguish from wild-type cells because their size decreased as a result of cell division. The first division of heat-arrested ccl cells after temperature down shift depended on the point in the cell cycle where the cells were arrested, and could occur between 1.5-6 hours (assume a 6.5 hour cell cycle) after the end of the heat treatment (Rasmussen and Berger, 1982). Therefore, isolation of all possible mutants before they recovered after an elutriation was unlikely due to the lengthy screening process. However, multiple elutriations allowed isolation of undiscovered potential mutants from previous elutriations. To assess the success of the mutant screen by elutriation, isolation of ccl cells from varying proportions of wild type mixed with ccl cell cultures by multiple elutriations was attempted. One hundred ccl cells were easily isolated in the later large cell fractions of the first extended elutriation of cultures which consisted approximately l .OxlO 6 cells of 10:1 and 100:1, wild-type to ccl cells (Table 1). Thus, no further extended elutriations were performed on these cultures. Multiple extended elutriations were next performed on a culture containing 5000:1, wild-type to ccl cells. The relative proportions of these two cell types were comparable to cell cycle mutant frequencies within a mutagenized culture (Peterson and Berger, 1976). A series of five extended elutriations were performed on a culture initially consisting approximately 300 ccl and 1.2xl0 6 wild-type cells. Large cells were only isolated after the third and fifth elutriations and not after every elutriation run because this allowed the ccl cells to increase in numbers before attempting to retrieve them. In Table 1, 33 and 83 ccl cells were retrieved from the third and fifth elutriations respectively, which together comprised 38% of the original ccl population. As expected, more ccl cells were isolated from the later fractions of the fifth elutriation. These results Table 1: Retrieval of ccl cells from mixed wild-type and ccl cultures. Mixed culture ratios Restrictive temperature (34) Total cells of wild type to c c l Wild type cd selected. Ten: one 8 118 126 Hundred:one 14 112 126 Five thousand:one-elutriation III 93 33 126 elutriation V 127 83 210 26 revealed an effective ability by the elutriation screening protocol to screen for sizable cells and regain a fraction of ccl cells originally present at low mutant frequencies. Isolation of cc mutants. Encouraged by the outcome of the above experiments, isolation of new cell cycle mutants by this elutriation screen was attempted. A wild-type culture containing approximately 1.2xl0 6 cells was divided equally into two smaller cultures and mutagenized with N'-methyl-N-nitro-N-nitrosoguanidine (lug/ml and 2ug/ml for 6 hours). A small fraction was set aside to serve as an untreated control. The cells were induced to undergo autogamy by starvation to achieve homozygosis of all gene loci (Sonneborn, 1970). The onset of autogamy was retarded in mutagen-treated cells when compared to the untreated culture. The untreated culture showed 77.1% of cells in autogamy the day following mutagenesis, while only 50.0% and 29.5% of 1 ug/ml and 2 ug/ml mutagen-treated cultures respectively were undergoing autogamy (Table 2). Delay in autogamy was presumably due to vegetative damage produced by the mutagen. Eventually, the proportion of mutagenized cells in autogamy reached levels comparable to the untreated controls four days after mutagenesis. One hundred percent of cells in autogamy was not achieved in the untreated controls because some cells may have undergone autogamy while the culture was raised prior to mutagenesis, and therefore these cells would be immature for autogamy. T o determine whether the mutagen was effective, the proportion of lethals in cell isolates from the two mutagenized cultures was measured. The cell isolates were selected after the cultures had undergone autogamy because during this time, micronuclear mutations induced were beginning to be expressed in the macronuclei. The higher mutagen dose damaged the cells to a greater extent as evident from the higher lethality rate (62.4%) than the 1 ug/ml mutagen-treated cells (46.2%) (Table 3). Consistent with this was the observation that a lower proportion of cells from the 2 ug/ml mutagen-treated culture Table 2: Effect of mutagen dose on induction of autogamy. Day after Culture type Percentage of cells mutagenesis in autogamy 1 untreated 77 1 mutagen-1 ug/ml 50 1 mutagen-2ug/ml 30 2 untreated 88 2 mutagen-1 ug/ml 66 2 mutagen-2ug/ml 68 3 untreated 72 3 mutagen-1 ug/ml 73 3 mutagen-2ug/ml 74 4 mutagen-1 ug/ml 84 4 mutagen-2ug/ml 81 28 Table 3: Percentage of cell lethality caused by mutagen dose. Culture type % lethality Adjusted % lethality % hit /genome untreated 9.52 0.00 0.00 mutagen-1 ug/ml 46.19 36.67 45.68 mutagen-2ug/ml 62.38 52.86 75.20 29 initiated autogamy following mutagenesis (Table 2). The untreated controls also exhibited some lethality although considerably less (9.5%) after autogamy. This was probably due to the presence of some deleterious mutations present in older cells prior to autogamy or perhaps by excessive starvation before testing. Adjusting for the nonmutagenic lethality in the controls, the 1 pg/ml and 2pg/ml mutagen dose produced 36.67% and 52.86% lethality respectively (Table 3). Using the formula P=e~u where P=l-observed lethality (%) and u represented the number of hits per cell, the probability of a mutagenic hit in a cell's genome was 45.68% and 75.20% by 1 pg/ml and 2ug/ml of mutagen respectively. The calculations were based on the assumption that the maximum number of hits that occurred in a cell's genome was one. Since the 2 pg/ml mutagen dose produced a high degree of exautogamous death (52.9%), taking into account the lethality rate present in the untreated controls and severe cellular damage, the culture treated with 1 pg/ml mutagen was chosen for cell cycle mutant isolation. The mutagenized culture was originally divided into three subcultures immediately after mutagenesis to facilitate complementation analysis if cell cycle mutants were recovered. Because multiple extended elutriations performed on one subculture required two weeks to complete, the two remaining subcultures became too starved and weak to be used even though they were stored at 17°C to decrease their growth and cell division. The mutagenized subculture was grown for four days after the maximal number of cells initiated autogamy to allow expression of recessive mutations before cells were subjected to the first extended elutriation. Separate elutriation runs were carried out on the mutagenized cell culture at four day intervals for a total of five elutriation runs. After each elutriation, the larger cells were selected from the latter fractions. Since the majority of these large cells were wild-type cells advanced in the cell cycle, cells that displayed unusual characteristics such as a darker appearance and/or an aberrant form were selected. The latter group included cells that exhibited abnormally round and elongated shapes. In addition, there were a few cells which possessed irregular shapes caused perhaps by a 30 Table 4: Cell types retrieved from elutriations of mutagenized culture. Dividers at Nondividers at restrictive temperature Total cells restrictive Elutriation temperature 1 2 3 4 5 selected 1 217 25 1 17 0 0 260 11 197 32 0 16 0 0 245 111 149 44 3 23 1 0 220 IV 141 49 10 50 0 0 250 V 60 60 8 114 3 7 252 Nondivider categories:(1) cells that died; (2) cells smaller than controls; (3) cells similar in size to controls; (4) cells of normal size with altered shapes; and (5) cells larger than controls. 31 failure to undergo cytokinesis after multiple rounds of nuclear division or a cytoskeletal defect. To further eliminate wild-type cells from the large cell pool, the selected cells were first placed together in a depression slide well for one to two hours before transferring the cells into isolates. This allowed any wild-type cells present to complete their cell cycle and proceed with division, thus appearing smaller than the remaining cells. A n average of 250 large isolates were selected from each elutriation run. The isolated large cells were permitted to recover and proliferate prior to testing their ability to divide at restrictive temperature. The remaining cells in the later fractions from each elutriation (about 5% of the culture) containing larger cells were incubated at 17°C for two days. The cells were stored at 17°C to lower wild-type cell proliferation as possible cell cycle mutants were recovering. This increased the probability of isolating cell cycle mutants in the next elutriation run because the proportion of possible cell cycle mutants was higher in the cell culture. The number of clones that failed to divide under restrictive conditions increased with each subsequent elutriation: 43, 48, 71, 109, and 192 nondividers at restrictive temperature were retrieved from the first, second, third, fourth, and fifth elutriations respectively (Table 4). This was consistent with the concept of increasing probability of recovery of cell cycle mutants in successive elutriations because they were allowed to multiply in numbers between elutriation trials. Conversely, the number of clones retrieved that were capable of cell division at restrictive temperature decreased after each elutriation run since the screening process selected against these cells. The nondividers at restrictive temperature were divided into five categories: (1) cells that died under restrictive conditions; (2) cells that were smaller than controls incubated at permissive temperature; (3) cells similar in size to controls; (4) cells of normal size with abnormal shapes and; (5) cells larger than controls. Because the first four categories did not show increased size after heat treatment, these cells were not cell cycle mutants, but probably carried mutations that affected growth and metabolism. The majority of these cells belonged to 32 the first and third categories while the remaining few cell lines were of the second and fourth categories (Table 4). Al l these cells were not maintained and eventually discarded. However, the fifth category of nondividers consisted of seven clones which were retained as putative mutants because they revealed some cell cycle mutant properties. The clones were all retrieved in the latter fractions from the fifth elutriation run and designated cc (cell cycle)3, cc4, cc5, cc6, ccl, cc8, and cc9. Cell cycle mutants arrest cell division at restrictive temperature. The cc mutants were tested repeatedly for their inability to divide accompanied by continued growth during heat treatment. This was accomplished by subjecting 10 newly divided cells from each cc mutant line to heat treatment for 18 hours. Repeated trials gave consistent results where cells of each cc mutant failed to divide under restrictive conditions but continued to grow and became considerably larger than their control counterparts incubated under permissive conditions (Table 5). With the exception of wild type, cc4, and cc6, fewer than the initial 10 cells used for heat treatment were retrieved after exposure to restrictive temperature ( 3 4 . 0 ° C ) in the remaining cc mutants. Since the cells missing ranged from 0-2 cells in each trial, failure to retrieve all the cells from the test tube or some lethality due to prolonged exposure to restrictive temperature might be the cause. cc4 and cc6 were leaky because some cell divisions occurred during heat treatment. In addition, cell growth was retarded in certain cc mutants (cc3, cc4, cc6, cc7, and cc9) at permissive conditions as evident from their lower number of cell divisions when compared to wild type. Because these cc mutants grew slowly at permissive temperature, the majority of them (cc4, cc6, cc7 and cc9) eventually perished. However, cc5 and cc8 exhibited growth comparable to wild type and complete division arrest at permissive and restrictive temperature respectively and they were studied further to examine the nature of their cell cycle blocks. 33 Table 5: Phenotypic expression of cell cycle mutants at permissive and restrictive temperature. Mutant TemDerature No. of cells No. of divisions wild type 27 72.67 2.86 34 85.00 3.09 cd 27 75.33 , 2.91 34 9.67 -0.05 cc3 27 46.00 2.20 34 9.00 -0.15 cc4 27 27.33 1.45 34 11.33 0.18 cc5 27 70.00 2.81 34 9.33 -0.10 cc6 27 31.67 1.66 34 10.33 0.05 ccl 27 20.33 1.02 34 8.00 -0.32 cc8 27 61.00 2.61 34 9.67 -0.05 cc9 27 17.33 0.79 34 9.00 -0.15 The initial cell number used was 10, and each value represented a mean of 3 trials. The number of cell divisions was calculated by the equation In (Nf/No)/ln 2, where Nf and No were the final and initial cell numbers respectively. 34 Transition point of cell cycle mutants. The next task was to determine whether cc5 and cc8 cells arrested at a specific point in the cell cycle under restrictive conditions. If they did, then cc5 and cc8 cells would possess a singular transition point, the time in the cell cycle when the temperature-sensitive gene product was no longer required in the subsequent cell division. Thus, the transition point marked the end of the temperature-sensitive period and was defined as the point in the cell cycle when 50% of the cells acquired the ability to divide following a shift to restrictive temperature (Frankel et al., 1980). The transition point of ccl was first determined by shifting synchronous cell samples rapidly to restrictive temperature ( 3 3 . 8 ° C ) at various times throughout the cell cycle to establish a control value. Fig.5 shows that the estimated ccl transition point from this study was 0.714 of the 2 7 ° C control cell cycle, similar to the results by Peterson and Berger, 1976. Transition points of 0.727 and 0.743 of the control cell cycle (5.6-6.6 hours) were obtained for cc5 and cc8 cells respectively (Table 6). These results indicate that similar to ccl, cc5 and cc8 transition points closely coincided with the Paramecium cell cycle control point responsible for commitment to cell division (Peterson and Berger, 1976: Rasmussen and Berger, 1984) and for the establishment of the timing of initiation of macronuclear D N A synthesis in the subsequent cell cycle (Ching and Berger, 1986). This suggest that the cc5 and cc8 gene products may be involved in the regulation of the Paramecium cell cycle. Temperature up shifts were not performed on wild type cells because of an absence of a transition point as they divided at the restrictive temperature. Temperature down shifts: effects on cell cycle duration. The temperature up-shift experiments did not indicate whether cc5 and cc8 cells were arrested only at a single point late in the cell cycle, or whether, like ccl the cells, they are arrested at any point prior to the transition point (Rasmussen and Berger, 1984). 35 Fig. 5: Transition Point of c d . Table 6: Transition point determination by temperature down shifts. Mutant Transition point-% of One SD control ceil cycle c d 0.714 0.000 cc5 0.727 0.030 cc8 0.743 0.016 37 To resolve this difference, synchronized newly divided cells initially exposed to restrictive temperature were down shifted to permissive temperature at various times throughout the cell cycle to determine the beginning of the temperature-sensitive period. If the temperature down shift occurred prior to the temperature-sensitive period, then the cell cycle duration would be unaffected. However, a cell cycle delay would be present if the down shift occurred after. Wild-type cells were first subjected to temperature down shifts to analyze whether they delayed their cell cycle as a result of the treatment. The restrictive temperature 3 3 . 8 ° C was used here instead of 3 4 . 0 ° C to minimize any heat shock effects present during the treatment and because this temperature completely arrested cell division in ccl, cc5, and cc8 cells (data not shown). Fig. 6 showed that wild-type cells down shifted earlier exhibited a slight delay (0.05 of the control cell cycle) in their cell cycle. This delay decreased as the cells were down shifted later and exposed longer to restrictive temperature. Eventually wild-type cells that were returned to permissive temperature after 0.70 of the 2 7 ° C control cell cycle divided earlier than the controls. This suggested that wild-type cells underwent a short temporary cell cycle delay when incubated initially at restrictive temperature. However, if the cells continued to be exposed to restrictive conditions after the cell cycle delay had ended, they accelerated their cell cycle progression and divided prior to the controls. When ccl cells were exposed to longer heat treatments prior to down shift to permissive temperature, their cell cycle duration was extended (Fig. 7). In agreement with Rasmussen and Berger, (1982, 1984), the extension of the cell cycle was found to be approximately equal to the heat treatment duration (Fig. 8). Since the cell cycle extension was caused by heat treatment contained wholly within the G l period, this implied that the beginning of the temperature-sensitive period in ccl cells occurred sometime in G l . This was confirmed by Rasmussen and Berger, (1984), who showed that the time elapsed between the end of heat treatment and the initiation of macronuclear S phase was equal to 38 Fig. 6: Temperature Downshift and One Hour Heat Pulse of Wild-Type Cells. ? 100 £ 80 S 7 0 1 60 I 50 TJ 40 | 30 8 20 10 0 10 20 30 40 50 60 70 80 90 100 Time of shift (% cell cycle control) 120 110 I 90 down shift 0 39 Fig. 7: Temperature Down Shifts of 4.0 5.0 6.0 7.0 8.0 9.0 10.0 11.0 Time after fission (hr) 40 the remainder of G l from the start of the heat treatment in ccl cells regardless of the heat treatment duration. Next, temperature down shifts were performed on cc5 and cc8 cells to determine whether they possessed similar temperature-sensitive starting points as ccl. Fig. 8 revealed that the beginning of the temperature-sensitive period in cc5 and cc8 were similar. Both mutants exhibited some delay in their cell cycle when exposed to restrictive conditions in G l , although this delay was slightly less than ccl (Fig. 8). However, similar to ccl, the cell cycle extension of cc5 and cc8 increased at comparable rates as the temperature down shifts took place later in the cell cycle (Fig. 8). These results indicated that cc5 and cc8 cells may possess similar temperature-sensitive periods as ccl which continued from G l to the point of commitment to cell division (Rasmussen and Berger, 1982, 1984). A n alternative possibility might be that cc5 and cc8 mutants contained two discrete temperature-sensitive points: one proximal to the G l / S boundary and the other around the G 2 / M transition. Thus, initial exposure to restrictive temperature would arrest the cells at the earlier temperature-sensitive point and delay the cell cycle roughly by the duration of heat treatment with the assumption that the cells were able to resume their cell cycle rapidly after temperature down shift. A possible method to distinguish the two possibilities was a double-shift experiment which involved the transfer of cells from permissive to restrictive temperature and then back again (Poodry et al., 1973). This temperature shock, when applied between the temperature-sensitive points, would not affect the cell cycle duration, but if superimposed within the temperature-sensitive period like that of ccl cells would result in an extension of the cell cycle. Fig. 6 showed the effect of one hour heat pulses at various times in the cell cycle on cell cycle duration in wild-type cells. The cell cycle delay increased as the heat pulses were applied later until late in the cell cycle. Heat pulses that occurred after this transition point did not extend the cell cycle duration. The problem encountered here was that the cell cycle delay as a result of the heat shock applied 41 Fig. 8: Temperature Down Shift of cc Mutants. 180 i — 100 I • • • • • i , • , • • , • I 0 10 20 30 40 50 60 70 80 90 100 Heat treatment (% cell cycle control) 42 towards the center of the cell cycle was considerable and comparable to the delay caused by one hour of heat treatment in the down-shift experiments (Fig. 6 and 8). Therefore, the cell cycle delay caused by both types of temperature-sensitive periods would be difficult to discriminate. For this reason, double-shift experiments were not performed on the cell cycle mutants. Genie relations of cc mutants. Table 7 showed the F l exconjugant phenotypes and postautogamous F2 segregation patterns obtained from the genetic analysis of the cc mutants. Proper cross fertilization occurred in all the matings because the behavioral paranoic marker segregated in a 1:1 ratio with wild type in the F2 progeny (Table 7; X 2 < X 2 0 05)- cc5 and cc8 cells were initially crossed to wild type to determine whether these mutations were dominant or recessive, and to acquire both mating types of each mutant which were used for later crosses. Both cc5 and cc8 mutations were recessive because all the F l exconjugants were phenotypically wild type. Furthermore, the cc trait segregated in a 1:1 ratio with wild type, as expected in the F2 postautogamonts. Since both cc5 and cc8 mutants possessed similar temperature-sensitive periods to ccl, the next step was to determine whether they were allelic or extragenic to ccl. This was accomplished by complementation crosses of ccl cells to both cc5 and cc8 mutants. The data revealed that cc5 and cc8 mutations complemented ccl because the F l exconjugants exhibited a wild-type phenotype. However, the F2 progeny from the two complementation crosses yielded different results. The F2 progeny of the ccl X cc8 mating showed the expected ratios of three temperature-sensitive cell cycle arrest (ccl/ccl;+/+, +/+;cc8/cc8, and ccl/ccl;cc8/cc8) to one wild-type phenotype (+/+;+/+). In contrast, the ccl X cc5 mating produced an additional class in the F2 progeny. These cells possessed an intermediate phenotype. They were larger and divided slightly slower than wild type at restrictive temperature. The F2 phenotypic ratios suggested that this cell 43 Table 7: Phenotypic ratios of F1 and F2 progeny from Genetic crosses. F1 Phenotype F2 Phenotype Mating cross ts+ ts pseudo Chi sq pa+ pa Chi sq c d X cc8L ts+ 14 28 1.14 16 26 1.93 c d X cc8R ts+ 12 30 0.13 24 18 0.60 c d X cc5L ts+ 15 40 25 2.50 39 41 0.01 c d X cc5R ts+ 12 51 6 16.82 31 36 0.24 cc8 X +/+L ts+ 17 21 0.24 21 17 0.24 cc8 X +/+R ts+ 22 17 0.41 20 19 0.00 cc5 X +/+L ts+ 21 16 0.43 22 15 0.94 cc5 X +/+R ts+ 21 14 1.03 17 19 0.11 cc8Xcc5IL ts 0 33 0.00 15 18 0.12 cc8 X cc5R ts 0 37 0.00 22 15 0.32 Chi square was calculated using Yates' correction for all crosses except cc5 X c d . The significant level excepted was P<0:05. All values fell within acceptable levels accept for cc5 X cc8R (ts+/ts). The null hypothesis of 1:1 was used in ts+/ts and pa+/pa frequencies for all crosses except 3:1 used in ts+/ts of cc8 X c d , 1:2:1 in +/ts/pseudo of cc5 X c d , and 1:0 in ts of cc8 X cc5. L and R designate the two progeny clones produced from a single mating pair. 44 class designated "pseudo" may represent double mutants (ccl/ccl;cc5/cc5) because approximately 25% of the F2 progeny constituted this class (Table 7; X 2 < X 2 0 0 5 ) . However, the frequency of "pseudo" progeny derived from the other parent of the mating pair (Table 7: cc5 X cc /R) was statistically different from 0.25 of the F2 generation (Table 7; X 2 > X 2 0 05). In addition, pooled data from both reciprocal crosses (cc5 X cclLlcclR) also failed to fit the 1:2:1 phenotypic ratio if the "pseudo class" was the ccSccl double mutant ( X 2 > X 2 Q 05; not shown). Because both reciprocal crosses were inhomogenous, the possibility of the "pseudo" class represented by either cc5 or ccl single mutants cannot be ruled out. Complementation tests between cc5 and cc8 showed that both mutants were allelic because all F l and F2 progeny ceased cell division under restrictive conditions. Together, these data indicated that the allelism of cc5 and cc8 was caused by two separate mutations at a single locus and not as a result of siblings from a common progenitor. 45 Discussion. This study develops a protocol to screen for Paramecium temperature-sensitive cell cycle mutants by size-selecting centrifugal elutriation and preliminary phenotypic characterization of putative cell cycle mutants retrieved from the protocol. The mutant screen is based on the observation that cell cycle mutants increase their cell mass after prolonged exposure to restrictive temperature. I was successful in isolating seven possible cell cycle mutants by using an elutriation centrifuge. These mutants cease cell divisions and continue to grow under restrictive conditions. Two of these mutants are allelic to one another and extragenic to ccl. The determination of their temperature-sensitive periods in the cell cycle by temperature shifts provides an insight in their gene expression and function. Phenotypic enrichment by centrifugal elutriation. Centrifugal elutriation is an effective method for separating a cell population into fractions of discrete size classes. This method has been used successfully in a variety of organisms (Gordon and Elliott, 1977; Fignor et al., 1983; Seyfert et al., 1984) including Paramecium (Adl and Berger, 1994; Tang and Berger, 1994) and is advantageous because fractionation of the cell population can be carried out in any media, thus minimizing disruption to growth. Currently, centrifugal elutriation is widely used in cell cycle research to obtain large numbers of synchronized cells for biochemical analysis (Schwob and Nasmyth, 1993; Tang and Berger, 1994). This process involves the collection of small G l daughter cells from the earliest fractions of an elutriated culture and allowing them to progress through the cell cycle. The synchronous cell population is sampled periodically for further analysis. A n alternate application of the elutriator is to isolate mutants abnormal for cell size. This is accomplished by selection of either small or large cells contained within the earlier and later fractions of a mutagenized culture respectively. 46 Seyfert et al., (1984) have used centrifugal elutriation to enrich for large Tetrahymena cells and were successful in isolation of temperature-sensitive size mutants. I have taken a similar approach to Seyfert et al., (1984) in an attempt to isolate temperature-sensitive Paramecium cell cycle mutants in this study but have made certain modifications to improve the screen. Large cell fractions from an elutriated culture can be obtained in two ways: (1) cells are first eluted at a specific flow rate while the remaining population containing the larger cells is washed out of the chamber after the centrifuge is shut off or; (2) successive fractions increasing in cell size are collected by raising the flow rate in small increments until the chamber is empty. The latter method was employed because cell fractions containing higher concentrations of large cells were obtained. In the segregated elutriated profiles of heat-treated wild-type and ccl cells, a cell fraction comprising of 78% and 20% of the original ccl and wild-type population would be acquired respectively if the elutriator rotor is stopped at the flow rate 75ml/min (Fig. 2). Thus, if the cell cycle mutant frequency is estimated at 0.02% (Peterson and Berger, 1976), a culture of 1.0X10 6 mutagenized cells subjected to this elutriation process would produce an enriched fraction of 156 possible cell cycle mutants and 2.0X10 5 normal cells (0.078%). However, if the same culture is elutriated in the other manner, then cell fractions collected at flow rates 77 to 83 ml/min would produce a fraction of 106 possible cell cycle mutants and 5.0X10 4 normal cells. Although the number of possible cell cycle mutants are reduced, these fractions are more enriched for this class of mutants (0.21%) because more normal cells are eliminated. Selection of large cells from these fractions would increase the chances of isolating cell cycle mutants. Another method that increases large cell enrichment is extended elutriations. The strategy here is to remove as many wild-type cells as possible by increasing the number of 25 ml aliquots collected at flow rates (59, 61, 63, and 65 ml/min) which elute and retain wild-type and ccl cells in the chamber, respectively. As a result, the proportion of wild-47 type cells are removed from the chamber at lower flow rates in greater than with segregated elutriations (Fig. 2 & 3). In addition, ccl cells are also eluted at lower flow rates in extended elutriations but their rate of removal from the chamber is lower than wild-type cells. Thus, cell fractions showing increases in concentration of large cells are attained. A 50 ml fraction collected at 67 and 69 ml/min immediately after the last of a series of 25 ml aliquots at 65 ml/min flow rate would comprise 55% and 3% of the original ccl and wild-type population respectively. If the starting population is assumed to be similar as above, then the concentration of possible cell cycle mutants would be 0.37%. The enrichment process of cell cycle mutants was estimated at 18.5 fold (0.02% to 0.37%) by using heat-treated ccl cells. A further enrichment of large cells can be accomplished by multiple elutriations. This technique increased the number and concentration of possible cell cycle mutants after each elutriation run because unselected large cells are allowed to multiply between elutriations. Therefore, the probability of isolating possible cell cycle mutants increases as more elutriations are performed. However, multiple elutriations could only be successful if the potential cell cycle mutants can recover from the heat treatment and proliferate after each elutriation run. The determination of the optimal maximum heat treatment required for the mutant screen is based on the assumption that cell cycle mutants possessed similar recovery rates to ccl. Results of ccl recovery rates indicate that the maximum duration of heat treatment possible without compromising recovery is about 14 hours (Fig. 1). Longer heat treatments decrease ccl recovery rates dramatically, at which a 75% reduction is seen after 24 hours at restrictive temperature (Fig. 1). Other cell cycle mutants may exhibit similar recovery kinetics to ccl cells, and therefore heat treatments considerably longer than 14 hours would negate the enrichment effects of multiple elutriations. As a result, 14 hours of heat treatments were used in the mutant screen. The enrichment capability of multiple elutriations can be assessed by the ability to retrieve ccl cells premixed with wild-type cells. Single elutriations are sufficient to recover 48 ccl cells present at concentrations of one and ten percent in a wild-type culture consisting of approximately l .OxlO 6 cells. The comparison of ccl cell enrichment and the achievable number of ccl cells retrieved is not practical at these ccl cell concentrations because the initial ccl cell population is too high. However, the relationship between these two factors can be examined and analyzed at ccl cell concentrations of 0.02% which is comparable to cell cycle mutant frequencies in Paramecium reported by Peterson and Berger, 1976. In order to calculate the ccl cell concentrations produced after each extended elutriation run, a number of assumptions is required. First, that an enriched cell fraction consisting of 4% and 73% of the starting wild-type and ccl population respectively was generated after each extended elutriation run by collecting eluted cells at flow rates of 67 ml/min and higher (Fig. 3). Second, that wild-type and ccl cells underwent an average of three and two cell divisions respectively in the time period from the end of each elutriation run to the beginning of heat treatment. Lastly, that wild-type cells divided twice while ccl cells were arrested during the heat treatment prior to the elutriation run. If the starting population consisted of l .OxlO 6 wild-type and 200 ccl cells, then the ccl cell concentration would increase to 0.37%, 0.83%, 1.90%, 4.33%, and 9.88% after the first, second, third, fourth, and fifth elutriations respectively. The number of ccl cells accumulates dramatically after five consecutive extended elutriations from 200 to 10614 cells. These figures indicate a tremendous enrichment capability for cell cycle mutants by multiple elutriations and therefore, a high probability in isolation of cell cycle mutants from a mutagenized cell population could be achieved by this technique. However, the results of the mixed culture containing 5000:1, wild-type to ccl cells retrieved after the third and fifth extended elutriations are much less than the ccl cell numbers estimated previously after each elutriation. Only 33 and 83 ccl cells were retrieved from the third and fifth elutriations respectively compared to 1244 and 10614 ccl cells hypothesized to be present after these elutriations. The low number of ccl cells isolated may be due to several reasons. Perhaps only a small fraction of ccl cells acquires 49 an increase in cell mass during heat treatment which is sufficient for discrimination from the remaining wild-type population by centrifugal elutriation. Previous studies by Berger, 1979, and Rasmussen and Berger, 1982 show that growth rates in Paramecium are dependent on gene dosage and cell mass. Coordinated increases in gene dosage and cell mass result in an increase in growth rate which, however, is proportional only to the relative magnitude of the lower of the two variables. In heat-treated ccl cells, the growth rate is proportional to the relative D N A content rather than cell mass because D N A synthesis is inhibited as cell mass continues to increase (Rasmussen and Berger, 1982). Therefore, the cell mass attained by ccl cells after heat treatment depends upon the cell cycle stage at the time of arrest at restrictive temperature. Cells arrested in G l would be smaller after the heat treatment than those cells arrested later in the cell cycle because they possess a lesser initial cell mass and a slower growth rate due to their small cell mass and D N A content. The largest ccl cells accumulated in a heat treated asynchronous population are those arrested just prior to the ccl transition point since their cell mass and D N A content is at a maximum amount. Thus, maybe only this minute subset of ccl cells is retrieved from the wild-type-cc7 mixed cultures. Slow growth of ccl cells during heat treatment occurred in some experiments as a result of medium contamination. The contamination consisted of a foreign bacterium that (outcompeted the regular food organism Klebsiella pneumoniae) and produced a substantial amount of bacterial slime. This often led to problems when growing mass cultures as the Paramecium cells were weakened. Since most of the experiments involved both wild-type and ccl cells, they were probably affected equally by the contamination. However, the retarded growth of ccl cells may have resulted in a lesser size differentiation from wild-type cells causing a lower yield of ccl cells retrieved from the multiple elutriation experiments. Another reason for the low yield of ccl cells could have been cell loss during the elutriation process. As ccl cells enlarge during heat treatment, the cortex, which functions 50 as a supportive flexible cell skeleton (Wichterman, 1986), undergoes increasing stress. Silver stained ccl cells exposed to restrictive temperature for prolonged time periods show that the somatic basal bodies within the cortex become further apart (observed unpublished results). This indicates that the complex fibrillar system of the cortex is being stretched and weaken, resulting in greater vunerability for cell lysis. Shearing forces that occur during centrifugation to concentrate and refeed cells as well as elutriations may destroy expanded ccl cells. Estimation of cell numbers prior and after each elutriation run reveals that roughly 10-20% of the original heat treated ccl population are lost. The nonsurviving cells are likely to be the larger ccl cells. The retrieval of fewer cc l cells than predicted in these experiments is probably due to all three reasons combined although it is uncertain which one is the dominant cause. Nevertheless, the ability to retrieve 38% of the original ccl population by multiple elutriations shows that this screening protocol is capable of isolating Paramecium cell cycle mutants. Mutagenesis and isolation of cell cycle mutants. The success of ccl cell retrieval from the mixed cultures from multiple elutriations made possible the isolation of potential cell cycle mutants from a mutagenized culture by the screening protocol. Seven putative cell cycle mutants that exhibited blockage of cell division and an increase in cell mass under restrictive conditions were isolated after five successive elutriations of a mutagenized culture. The observation that all the cc mutants were retrieved after the fifth elutriation (Table 4) was significant because the probability of isolating possible cell cycle mutants increased after each elutriation. In addition, the number of nondividers retrieved also increased as more elutriations were performed (Table 4). This indicates that mutants that cease cell divisions at restrictive conditions due to either a metabolic or a cell cycle defect are able to recover and proliferate inbetween elutriation runs. 51 The cell cycle mutant frequency in Paramecium obtained by Peterson and Berger, 1976 is approximately 0.02%. If this frequency is assumed to be accurate, then approximately 120 potential cell cycle mutants should be present among the 6.0X10 5 mutagenized paramecia used in this study: However, only seven cell cycle mutants were isolated after five elutriations, much fewer than the estimated number above. A possible reason for this is that most Paramecium cell cycle mutants require longer recovery periods after heat treatment than predicted. The duration of heat treatment used in this screen was determined by the ability of ccl cells to recover. Rasmussen and Berger (1984) have shown that, unlike other cell cycle mutants, ccl cells are able to overcome their arrest in macronuclear D N A synthesis and continue the remainder of their cell cycle quickly within 20 minutes after down shift to permissive conditions. Longer recovery times of other cell cycle mutants would negate the selective advantage of multiple elutriations. Cell cycle mutants possessing poor recovery following heat treatment would not multiply quickly prior to each elutriation run and as a result, the probability of retrieving them is reduced. Because these mutants are present at lower numbers, any loss of these cells during the elutriation process would impede their isolation much more than cell cycle mutants with better recovery. However, underestimation of recovery time is not sufficient to account for the low number of cell cycle mutants retrieved. This is supported by the low yield of ccl cells retrieved from multiple elutriations of wild-type-cc/ mixed cultures. The reasons discussed above, such as cell loss during the elutriation process and a small subset of cells that actually acquire a significant increase in cell mass recognizable by the elutriator, could also contribute to the reduction in cell cycle mutant isolation. The low yield of cell cycle mutants may also be caused by an inadequate number of elutriations performed. The results from the wild-type-cc/ mixed culture elutriations show that the number of ccl cells retrieved increased as more elutriations were performed (Table 1), although their numbers were much lower than predicted. This suggests that 52 accumulation of ccl cells is slower during the elutriation process due to the probable causes discussed previously and that additional elutriations should increase the yield of ccl cells retrieved. This phenomenon may also exist in the mutagenesis screen as all the cc mutants were isolated after the fifth elutriation. However, since a wide variety of cell cycle mutants were presumably present within the mutagenized culture, there are certain mutants that would remain undiscovered regardless of the number of elutriations performed. Such mutants may possess microtubule defects at restrictive temperature and fail to recover at all after temperature down shift as a result of chromosome missegregation. Several mutants of this type have been discovered in Saccharomyces cerevisiae by Winey et al. (1991, 1993), Page and Synder (1992), and Saunders and Hoyt (1992) exhibiting abnormal spindle pole body duplication and segregation, and loss of mitotic spindle integrity caused by nonfunctional kinesin-related proteins. Thus, these mutants would be lost and not isolated during multiple elutriations. Although additional elutriations may have increased the yield of newly discovered cell cycle mutants, only five elutriations were performed on the mutagenized culture. One of the primary objectives of this study was to isolate one or two new cell cycle mutants instead of as many as possible because preliminary characterization of the latter would be too time consuming. The seven putative cell cycle mutants retrieved after the fifth elutriation fulfilled this objective. Perhaps future mutagenesis could employ more elutriations to isolate many new Paramecium cell cycle mutants. However, this approach creates an additional problem as siblings become more easily isolated than new cell cycle mutants with increasing numbers of elutriations. Therefore, a trade off exists where too few elutriations would result in isolation of a very small fraction of possible cell cycle mutants within a mutagenized culture, while too many elutriations cause a high number of siblings retrieved, hampering progress in analysis. The optimal number of elutriations would depend upon the relative proportions of cell cycle mutants with good and poor recovery. If the cell cycle mutants possess predominantly good recovery rates like ccl 53 cells, then the optimal elutriation number would be lower than if most of the mutants show poor recovery. Temperature-sensitive periods of cc5 and cc8. Analysis of cc5 and cc8 temperature-sensitive periods provides information on the possible functions of these genes. The transition points of cc5 and cc8 are closely associated with the point of commitment to cell division in Paramecium (Table 6). Although this suggests that cc5 and cc8 genes may function in a cell cycle event prior or up to the point of commitment to cell division, the possibility that they function in an event after commitment to division cannot be ruled out. Mutants have been discovered by Nurse et al. (1976) that possess a transition point considerably before the terminal phenotype. This phenomenon can be caused by the defective synthesis of a protein made early and used later in the cell cycle (Hartwell, 1978). In order to distinguish between these two possibilities, the terminal phenotype and its temporal location relative to the transition point of cc5 and cc8 must be determined. First the terminal phenotype of both mutants must be characterized. This can be accomplished by cytological staining of asynchronous cells exposed to restrictive temperature for at least one control cell cycle duration to detect for aberrant cytoskeletal structures and nuclear division. The terminal phenotype can then be mapped to known cell cycle landmarks in Paramecium such as the stages of oral morphogenesis and micronuclear mitosis (Adl and Berger, 1991, 1992), and compared to the point of commitment to cell division. The nature of the terminal phenotype also gives an indication of the cc5 and cc8 gene functions. The temperature down-shift data reveals that the start of the temperature-sensitive period of cc5 and cc8 may occur in G l because a cell cycle delay is present when the cells are heat treated early in the cell cycle (Fig. 8). The cell cycle delay as a result of the earliest down shift is slightly less than that in ccl which suggests that the temperature sensitivity of cc5 and cc8 may begin later than ccl in G l . The cell cycle extensions 54 increase at a similar rate as the temperature down shifts occur later in all three cc mutants (Fig. 8). These results indicate one of two possibilities: (1) cc5 and cc8 cells may possess a long temperature-sensitive period from G l to the point of commitment to cell division such as ccl cells or; (2) cc5 and cc8 cells may contain two discrete temperature-sensitive points, one located early and the other one late in the cell cycle similar to cdc2 mutants in S. pombe (Nurse and Bissett, 1981). To resolve which situation occurs in cc5 and cc8, a double-shift experiment can be performed. A heat pulse applied approximately in the middle of the cell cycle would cause a cell cycle delay at least the duration of the heat pulse in the first scenario and little or no delay in the latter. However, the amount of cell cycle delay measured from double-shift experiments is sometimes difficult to interpret for two reasons. First, the cell cycle delay caused by the double shifts of wild-type cells increases as the heat pulses are applied later in the cell cycle until proximal to the point of commitment to cell division (Fig. 6). After this transition point, the heat pulses have no effect on the cell cycle duration. The pattern of the cell cycle delays as a result of the heat pulses resembles the cell cycle delay caused by heat shock (Zeuthen and Rasmussen, 1971; Polanshek, 1977). Second, distinct cell cycle mutants are most likely to recover with different kinetics upon return to permissive conditions (Pringle, 1978). These two factors create difficulty in accurate measurement of the cell cycle delay caused exclusively by the heat pulse. One way to further characterize the phenotype of cc5 and cc8 cells is to examine whether these cells synthesize D N A when they are shifted up to restrictive temperature during S phase. Cells that possess a similar temperature-sensitive period to ccl would inhibit D N A synthesis when exposed to restrictive conditions, but the contrary would occur if the temperature sensitivity existed at two discrete points early and late in the cell cycle. Labelling of synchronous Paramecium cells shifted to restrictive temperature at various points in the cell cycle with [ 3H] thymidine-labelled bacteria and autoradiography 55 would detect the presence of D N A synthesis in these mutants during S phase (Berger, 1971). Genie relations between cc mutants. Complementation tests reveal that cc5 and cc8 are allelic to each other and extragenic to ccl (Table 7). The allelism of cc5 and cc8 is probably caused by two distinct mutations in a single locus rather than siblings from one mutation. This is supported by dissimilar F2 phenotypic ratios observed in the cc5/cc8 X ccl crosses (Table 7). A n additional class of F2 postautogamonts displaying an intermediate phenotype of ccl and wild-type cells is present in the cc5 X ccl but not the cc8 X ccl matings. These cells constitute approximately 25% of the F2 progeny from the pooled data. Two possible explanations may account for these results. First, the "pseudo" cells may be cc5ccl double mutants because this intermediate phenotype is unique since it does not resemble the other three classes of the F2 progeny (cc5/cc5;+/+, +/+;ccl/ccl, and +/+;+/+). Alternatively, the "pseudo" cells may represent leaky cc5 and/or ccl single mutants due to an inadequate test temperature that failed to inhibit cell division completely. In this case, the "pseudo" cells are likely to be cc5 mutants because they exhibit higher temperature tolerance than ccl cells (observed unpublished data). A method that is able to discriminate between these two possibilities is a back cross that involves mating of the "pseudo" lines with both cc5 and ccl cells. If the "pseudo" cells are ccSccl double mutants, then a mating of "pseudo" cells with cc5 and cc7 mutants would produce an F2 generation of 1:1 "pseudo" (cc5/cc5;ccl/ccl) to temperature-sensitive (cc5/cc5;+/+ or +/+;ccl/ccl) cells. The F l progeny (cc5/cc5;ccl/+ or cc5/+;ccl/ccl) may be "pseudo" or temperature-sensitive depending on the amount of cc5 and ccl gene products required for the "pseudo" expression. In contrast, if the "pseudo" cells represented cc5 mutants, then a cross of "pseudo" with cc5 cells would produce temperature-sensitive F l and F2 progeny (all cc5/cc5), while mating "pseudo" with ccl cells would produce a wild-type F l generation 56 (cc5/+;ccl/+) and a 1:2:1 phenotypic ratio of "pseudo" (cc5/cc5;ccl/ccl), temperature-sensitive (cc5/cc5;+/+ and +/+;ccJ/ccJ), and wild-type (+/+;+/+) F2 progeny. The appearance of the "pseudo" cells in the F2 progeny may also be the result of phenotypic lag in the postautogamonts of the F l heterozygotes. Following loss of the wild-type allele from the macronucleus, the parental phenotype persists for 6-12 cell cycles due to continued gene activity in macronuclear fragments carrying the wild-type allele (Berger, 1976). The few cell lines exhibiting the "pseudo" phenotype from the F2 progeny of the other parent seem to suggest this. In addition, the cc8 X ccl cross did not produce any "pseudo" clones in the F2 generation. However, this scenario is unlikely because the F2 progeny were tested in the water bath after they had undergone more than 12 fissions following autogamy and so phenotypic lag should have been absent. Therefore, no "pseudo" cells should be seen because these cells would be temperature-sensitive. If the "pseudo" cells are assumed to be ccSccl double mutants, then this indicates that the presence of both mutated cc5 and ccl gene products is able to suppress the cell cycle arrest phenotype of cc5 and ccl single mutants. This suggests that the cc5 and ccl products may function in a common cell cycle process such as macronuclear D N A synthesis by interaction with one another. Inhibition of this essential interaction would occur in cc5 and ccl single mutants resulting in cell cycle arrest, while cc5ccl double mutants would allow this interaction to occur. Supportive evidence for this model includes the isolation of several ccl extragenic suppressors indicating that the ccl+ gene product functions as a multicomplex (unpublished data). In addition, one of the ccl extragenic suppressors has been shown recently to have the ability to suppress cc8 mutants. This observation demonstrates that the cc5/cc8 gene product interacts with the ccl multicomplex. A n important question that remains elusive is what are the functions of the individual participants in this multicomplex? Construction of mutants that possess different combinations of these multicomplex proteins by various crosses and their analysis to search for distinguishing phenotypes would assist in answering this question. 57 In summary, this study has shown that centrifugal elutriation is capable of large cell enrichment to a degree that is effective for isolating temperature-sensitive cell cycle mutants in Paramecium. This is beneficial to cell cycle research because discovery of new mutants permits identification of key participants in cell cycle regulation. Because molecular research in Paramecium is limited, determination of the temporal periods of temperature sensitivity in the cell cycle by temperature shifts in these mutants allows an alternative method to acquire information on their gene expression and function. However temperature shifts are not enough because they have their limitations. 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