I (ALLEN 1965; GIBSON 1970). In Tetrahymena pyriformis, syngen 1, a genetically

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1 SHORT PAPERS MUTANT SELECTION IN TETRAHYMENA PYRIFORMIS PETER S. CARLSON' Department of Biology, Wesleyan University, Middletown, Connecticut Received June 18, 1971 NTRACLONAL variation is a well documented phenomenon of ciliate genetics I (ALLEN 1965; GIBSON 1970). In Tetrahymena pyriformis, syngen 1, a genetically heterozygous clone will segregate subclones which are phenotypically homozygous for each of its two alleles (c.f. ALLEN 1965; ALLEN and NANNEY 1958). Despite the appearance of functional homozygosity in the macronucleus, the micronucleus maintains its genetically heterozygous constitution. This paper describes a mutant selection scheme which takes advantage of heterozygote resolution in the ciliate macronucleus to generate homozygous mutant organisms. The experiments are based on the following design. Several populations of T. pyriformis, syngen 1, of different mating types are treated with a mutagen and then mated. The micro- and macronucleus of some of the exconjugants will be heterozygous for mutations. The exconjugant populations are forced to undergo a number of fissions to permit the macronucleus of individuals in the population to become functionally homozygous for a mutant trait. The entire population is then subjected to a selective screen such that only defined mutant types are able to survive. Survivors will possess a functionally homozygous mutant macronucleus and a genetically heterozygous micronucleus. By subsequent crosses the mutant allele can be recovered for analysis in a homozygous condition in both the micro- and macronucleus. Further crosses also verify that the altered phenotype is due to a genetic lesion rather than to a nonnuclear event or to a stable physiological alteration (dauermodification). MATERIALS AND METHODS Three stocks of Tetrahymena pyriformis, syngen 1, which were obtained from DR. DAVID NANNEY, were used exclusively in this work. These are stock A K, mating type V; stock B , mating type 111; and stock B , mating type VII. Cultures were grown on 1% proteosepeptone medium at 22 C. Axenic conditions were maintained throughout the course of the work. Alcohol dehydrogenase activity was determined by the method of RACKER (1955). Protein was determined by the method of LOWRY et al. (1951). Ethyl methanesulfonate and fluoroacetic acid were obtained from Pfaltz and Bauer, Inc. Caffeine was obtained from Aldrich Chemical Co., and allyl alcohol from Eastman Organic Chemicals. 14C-labeled allyl alcohol was synthesized by the method of KAMM and MARVEL (1921) from Glycerol-2-14C obtained from New England Nuclear. Methyl-14Gcaffeine was also procured from New England Nuclear. * Present addiess Department of Biology, Brookhaven National Laboratory, Upton, N.Y Genetics 69: October, 1971.

2 262 P. S. CARLSON RESULTS AND DISCUSSIONS The Populations: T. pyriformis, syngen 1, of three different mating types (111, V, and VII) were treated with.25% ethyl methanesulfonate for 1 hr. 1.5 X 1 O6 viable cells of each mating type were then pooled under conditions for conjugation. Over 93% of the cells formed conjugating pairs, with a total of 2.2 x 1 O6 conjugating pairs per population. Exconjugants were subsequently cultured for approximately 120 fissions before being subjected to the selective screen. Two different populations, numbxs 1 and 2, were produced by this procedure. Three chemicals were used as selective screens: fluoroacetic acid (F.A.A.), allyl alcohol (A.A.), and caffeine (C.). No clones resistant to normally toxic levels of F.A.A. were recovered. One clone resistant to high concentrations of A.A. was obtained as were two clones resistant to usually toxic levels of C. An analysis of the A.A. and C. resistant clones forms the remainder of this report. Preliminary attempts to select auxotrophic mutants using the 5-bromo-deoxyuridine technique (PUCK and KAO 1967) were unsuccessful. The allyl alcohol resistant clone: Allyl alcohol has been shown by MEGNET (1965) to be an effective selective agent in yeast for mutants which lack alcohol dehydrogenase (ADH). A.A. kills wild-type cells, but is not lethal to mutant individuals. Wild-type Tetrahymena are normally killed within 12 hr by 250 mm A.A. A clone was isolated from population 2 whose viability was not affected by normally toxic levels of A.A. The following series of experiments, based on the predictions of Mendelian genetics, were performed to determine if the resistance is due to a nuclear, a nonnuclear, or a physiological alteration. Recall that cells of the resistant clone are expected to contain a heterozygous micronucleus. Cells of the resistant strain were mated with wild-type individuals. 32 viable conjugating pairs were isolated and exconjugants were tested for resistance to A.A. after 10 and again after 70 fissions. All 32 isolates were sensitive to A.A. after 10 fissions. After 70 fissions, 17 of the 32 isolates remained sensitive to A.A. while 15 isolates contained cells which were resistant to the chemical. The 15 isolates which contained resistant cells were presumably heterozygous for the mutant trait and segregated phenotypically homozygous cells over c? number of fissions. The 17:15 ratio corresponds to an expected 1:l Mendelian ratio, and indicates that the resistance factor is recessive. Cells of the 15 resistant isolates were used for further crosses to the original resistant clone. Of 148 viable conjugating pairs recovered from such matings, 33 were resistant to A.A. after 10 fissions. The ratio of 115:33 corresponds to an expected 3:l Mendelian ratio. One further expectation of a Mendelian trait is that 2/3 of the 115 sensitive lines should give rise to resistant cells after a number of fissions. To test this expectation 47 of the 115 lines were carried through 70 fissions. Tests after this period indicated that 27 of the lines contained resistant cells. The 27/47 ratio approximates the expected 2/3. Further evidence for the genetic character of the resistance comes from matings

3 MUTATION IN TETRAHYMENA 263 between the original resistant clone and several of the 17 sensitive lines of the initial cross. In these matings none of the isolates were resistant after 10 fissions and approximately 1/2 of the isolates contained some resistant cells after 70 fissions. Hence, the resistance trait must be determined by a recessive, nuclear gene. Any physiological or nonnuclear explanation finds no basis in the mode of resistance transmission. The following work was done to determine the biochemical basis of the resistance phenotype. Resistance to A.A. may be conferred by a loss of ADH activity. Assays of ADH in wild type and homozygous mutant clones did not demonstrate significantly different amounts of enzyme activity between the two. Hence, absence of ADH activity is not the biochemical basis of A.A. resistance. I4C labeled A.A. was used to examine the level of accumulation of the chemical within the organism. Experiments demonstrated that mutant cells do not accumulate A.A. at the same rate as wild type. After a two-hour exposure to labeled A.A. the wild-type cells contained 9 times the level of A.A. found in mutant cells. Hence, A.A. resistance appears to be due to an altered level of accumulation of the chemical. The accumulation of glycerol and ethanol were unaffected by the mutation. An analysis of intraclonal variation was completed using 3 heterozygous clones produced by mating homozygous mutant cells with wild-type individuals. Cells resistant to A.A. begin to appear after 50 to 60 fissions. Following the rationale of ALLEN and NANNEY (1958), the average rate of segregation of resistance types was calculated to be.0126 per fission. (The value is corrected for the fact that only 1/2 of the stable types can be scored for the resistance phenotype.) This value is close to the expected value of.0113 (c.f. ALLEN and NANNEY 1958). The cafleim resistant clones: Wild-type Tetrahymena are normally killed by 10 mm caffeine. Two caffeine resistant clones were recovered, one clone from each of the two populations. Both clones were able to survive and grow in 10 mm caffeine and survive for several days in concentrations up to 25 mm. The resistant clones are referred to as clone 1 and clone 2. The following crosses, based on the previously outlined rationale, were completed to verify that the resistance of the clones was due to a Mendelian factor. Cells from clone 1 were mated to wild-type cells, and 12 conjugating pairs were isolated. After 10 fissions none of the exconjugant lines contained cells which were caffeine resistant. After 70 fissions, 4 of the lines had segregated resistant cells. Cells of one of the lines which contained resistant individuals were mated with cells of clone 1. Of 12 isolated conjugating pairs, 4 were resistant to caffeine after 10 fissions. Of the 8 isolates which were sensitive to caffeine after 10 fissions, 6 lines contained resistant cells after 70 fissions. 2 isolates did not contain any resistant cells after 70 fissions. The 4:6:2 ratio approximates the expected 1:2:1 Mendelian outcome. Cells of one of the eight lines of the initial cross which did not contain resistant individuals were mated to cells of clone 1. Of 9 isolated conjugating pairs, none were caffeine resistant after IO fissions, and only 3 contained resistant cells after 70 fissions. The resistance of clone 1 must be determined by a recessive Mendelian genetic factor.

4 264 P. S. CARLSON Cells from clone 2 were taken through an equivalent series of crosses as outlined for clone 1. Clone 2 cells were mated with wild-type cells and 13 conjugating pairs were isolated. Of the 13 isolates, none were resistant to caffeine after 10 fissions. Five isolates contained resistant cells after 70 fissions. Cells of one of the lines which contained resistant individuals were mated with clone 2 cells. Of the 12 isolated conjugating pairs, 2 showed resistance to caffeine after 10 fissions, 7 isolates contained resistant cells after 70 fissions, and 3 isolates were composed of totally sensitive cells after the 70 fissions. Hence the resistance of clone 2 must also be a recessive Mendelian gene. To determine if the two mutants were allelic, cells homozygous for the clone 1 resistance mutation were mated to cells homozygous for the clone 2 resistance. 3 conjugating pairs were isolated. None of these isolates showed resistance to caffeine after 10 fissions. Hence, the mutations are not allelic. The biochemical basis of the resistance phenotypes was examined by analyzing the uptake of I4C-labeled caffeine. Cells homozygous for the clone 1 resistance accumulate only 1/3 of the level of caffeine found in wild-type cells after a 1/2 hr pulse. Cells of clone 2 show no difference in level of accumulation from wild type. Hence, the clone 1 mutation appears to affect caffeine accumulation. The physiological basis of the clone 2 mutation remains unidentified. Intraclonal variation was examined in clones heterozygous for the mutant alleles. Clones heterozygous for the clone 1 resistance first contained resistant cells after 20 to 30 fissions. The average rate of segregation of resistance types for the clone 1 mutation was calculated to be.0097 per fission. In clones heterozygous for the clone 2 mutation, resistant cells began to appear after 40 to 50 fissions. The rate of segregation was determined to be.0109 per fission. The results of this work demonstrate that it is possible to select phenotypically mutant individuals from genetic heterozygotes after macronuclear segregation. The technique should be useful for recovering mutant types which can be separated from wild-type cells by chemical, visual, mechanical or environmental means. The resistance mutants provide verification of the behavior of the macronucleus during somatic fissions, and suggest that macronuclear segregation may be a phenomenon common to many genetic loci in ciliates. It is a pleasure to thank Drs. R. F. KIMBALL, D. L. NANNEY, M. GOROVSKY, and J. WOLFE for their help. ALLEN, S. L., : LITERATURE CITED Genetic control of enzymes in Tetrahymena. Brookhaven Symp. Biology ALLEN, S. L. and D. L. NANNEY, 1958 An analysis of nuclear differentiation in the selfers of Tetrahymena. Am. Naturalist 92: GIBSON, I., 1970 Interacting genetic systems in Paramecium. Advan. Morphogenesis 8: KAMM, 0. and C. S. MARVEL, 1921 Allyl Alcohol. Organic Synthesis 1:

5 MUTATION IN TETRAHYMENA 265 LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, 1951 Protein measurement with the folin phenol reagent. J. Biol. Chem. 193: MEGNET, R., 1965 Alkoholdehydrogenasemutanten von Schizosacchmomyces pombe. Path. Microbiol. 28: 5&57. PUCK, T. T. and F. T. KAO, 1967 Genetics of somatic mammalian cells. V. Treatment with 5-bromodeoxyuridine and visible light for isolation of nutritionally deficient mutants. Proc. Natl. Acad. Sci. USA. 58: RACXER, E., 1955 Alcohol dehydrogenase from Baker s yeast. pp In: Methods in Enzymology. Vol. 1. Edited by S. P. COLORICK and N. 0. KAPLAN. Acad. Press, New York.