and KENNEDY 1973; FRANKEL - Genetics

MUTANTS OF TETRAHYMENA THERMOPHILA WITH TEMPERATURE-SENSITIVE FOOD VACUOLE FORMATION.

I. ISOLATION AND GENETIC CHARACTERIZATION

PETER B. SUHR-JESSEN1 AND EDUARDO ORIAS

Department of Biological Sciences, University of California at Santa Barbara, Santa Barbara, California 93106

Manuscript received July 27, 1978 Revised copy received January 30,1979

ABSTRACT

Germ-line mutants have been isolated in Tetrahymena thermophila that have recessive, temperature-sensitive defects in phagocytosis. Nitrosoguanidine- mutagenized cells were induced to undergo cytogamy, and clones were isolated that were unable to form food vacuoles after two days of growth at 39". Most of the mutants belong to a single complementation group, designated uacA. They have defects in oral development-not in phagocytosis per se-that are undetectable under light microscopy. One fertile mutant, phenotypically in- distinguishable from the vacA group, has its uac mutation(s) restricted to the macronucleus, and it is a heterokaryon for two other markers. This clone probably resulted from a failure of the two gametic nuclei to fuse after nor,mal exchange. Two additional mutants were studied, but their sterility prevented a full genetic analysis. One of these clones has a rudimentary oral apparatus and defective contractile vacuole pores; both defects may be determined by the same mutation. The other clone has a structurally normal oral apparatus and may be defective in phagocytosis per se.-The induction and characterization of germ-line mutations that affect oral development open the way for the genetic dissection of the morphogenesis of a complex eukaryotic organelle, and make available additional useful mutants for the study of nutrition and trans- membrane active transport.

HE oral apparatus (OA) of Tetrahymena is located on the anterior ventral Tsurface of the cell (FURGASON 1940; ELLIOTT and KENNEDY 1973; FRANKEL and WILLIAMS 1973). Studies using light and electron microscopes 01 in situ and isolated OA's reveal that this organelle is about 11 pm long and 6 piii wide and consists of three groups of ciliary rows (which contain at least four classes of cilia) surrounded to the right and posterior by an undulating membrane (STEWART 1977; BUHSE, STAMLER and CORLISS 1973; FORER, NILSSON and ZEUTHEN 1970; NILSSON and WILLIAMS 1966; SATTLER and STAEHELIN 1974, 1976). The buccal cavity leads to the cytopharynx where food vacuoles with average diameters of about 5pm are sequentially formed and released into the cytoplasm (NILSSON 1972; CHAPMAN-ANDRESEN and NILSSON 1968; NILSSON and WILLIAMS 1966; SATTLER and STAEHELIN 1976).

1 Present address: Biological Institute of the Carlsberg Foundation, Tagensvej 16, DK-2200 Copenhagen N, Denmark.

Genetics 92: 1061-1077 August, 1979.

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1062 P. B. SUHR-JESSEN AND E. ORIAS

An orderly sequence of six morphological stages of oral development during the last 90 minutes prior to cell division results in a functional OA for the POS- terior daughter cell ( FRANKEL 1962; BUHSE, STAMLER and CORLISS 1973; SUHR- JESSEN, STEWART and RASMUSSEN 1977). Proteins are the major component of the OA, comprising about 2.3% of the total cell protein (ZELNIS, cited by FRANKEL and WILLIAMS 1973). The major OA proteins are tubulins, but other proteins are present (GAVIN 1974; VAUDEAUX 1976). In view of the structural, functional and developmental complexity of the OA, one would expect that many gene products must be required for its normal activity.

One mutant with temperature-sensitive development of the OA was previously isolated in inbred stain D (ORIAS and POLLOCK 1975). This mutant is not very useful genetically because its mutation is restricted to the macronucleus ( SILBER- STEIN, ORIAS and POLLOCK 1975). Nevertheless, this mutant was used to demonstrate that food vacuole formation is dispensable for cell growth and division if the ordinary gowth medium is supplemented with high concentrations of Fef3, CufP and folinic acid (ORIAS and RASMUSSEN 1976).

A genetic defect of OA morphogenesis, spontaneously occurring in inbred strain D of T. thermophila, has been reported by KACZANOWSKI (1975, 1976). Crosses between inbred strains D and A revealed a single, incompletely pene- trant, genetic difference resulting in supernumerary oral membranelles. The level of expression of this defect can be increased by prolonged starvation.

We have now undertaken a genetic dissection of the events leading to the development of a functional OA and the formation of food vacuoles. This was facilitated by the recent discovery that cytogamy can be efficiently induced by a properly timed osmotic shock (ORIAS, HAMILTON and FLACKS 1979). We report here the induction, isolation and genetic characterization of a collection of mutant clones with temperature-sensitive defects in food vacuole formation in T. thermophila. Their morphological and physiological characteristics will be reported elsewhere ( SUHR-JESSEN and ORIAS, in preparation). One of the mutants turned out to be a heterokaryon that probably originated from a rare failure of the gametic nuclei to fuse after normal exchange during conjugation.

MATERIALS A N D METHODS

Strains Inbred strain BIII of Tetrahymena thermophila (NANNEY and McCoy 1976) was obtained

from D. L. NANNEY and employed throughout this study. Functional heterokaryons derived from inbred strain B (BRUNS and BRUSSARD 1974) were obtained from P. J. BRUNS (Table 1).

TABLE 1

Functional heterokaryons employed

Clone Genetic descriDtion Suurce

CU324 Mpr-l/Mpr-1 (6-mp-S, IV) P . J. BRUNS CU329 ChzA2/ChzA2 (cycl-S, 11) P . J. BRUNS CU330 ChzA2/ChzA2 (cycl-S, IV) P . J. BRUNS

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FOOD VACUOLE-LESS M U T A N T S OF TETRAHYMENA 1063

They were used for the mutant isolations and, together with wild-type strain BIII, for the genetic characterizations. A collection of tester strains of mating-types I1 to VI1 was used for mating-type tests.

Media Nutrient media: (to which 250 yg per ml each of penicillin G and streptomycin sulfate

(Sigma) was added) : PPF: Autoclaved 2% proteose peptone broth (Difco) supplemented with salts (KIDDER and DEWEY 1951). CHX: PPF supplemented with 15 pg per ml cycloheximide (Sigma). MP15: PPF supplemented with 15 pg per ml 6-methylpurine (Sigma). MP75: PPF supplemented with 75 pg per ml 6-methylpurine. EPP: Autoclaved 2% proteose peptone broth enriched with 2 m ~ sodium citrate, 1 mM FeCl,, 30 p~ CuSO, and 1.7 PM folinic acid (ORIAS and R~SMUSSEN 1976). INK: EPP supplemented with 0.4% (v/v) india ink (Higgins).

Staruation media: (1) Dryl's salt solution (DRYL 1959). (2) 10 miu Tris-HC1 (pH 7.5) ( ORIAS and BRUNS 1976).

Mating test medium: 2% bacterized peptone (ORI.4S and FLACKS 1973).

Maintenance of stocks

test tubes containing 2 ml of EPP and diluted 1000-fold each week.

Standard axenic cross Early stationary cultures grown in PPF medium at 22 or 30" were washed into 10 ml of

starvation medium at a cell density of about 105 cells per ml and starved overnight in large petri plates at the same temperature. Five ml of each of two strains were then mixed in large petri plates. After seven to ten hr at 30", or 16 to 20 hr at 22", single pairs were isolated at room temperature into drops of PPF in a large petri plate using a manually controlled micropipette under a dissection microscope. The plates were incubated for at least three days at 22 or 30". In some experiments, exconjugants were separated as described by ORIAS and FLACKS (1973).

Phenotype tests

The "drop" cultures were tested for resistance to cycloheximide and 6-methylpurine, for food vacuole formation and for maturity, as described below.

Drug tests: Drop cultures were replicated to CHX and MP15 medium at 30" and were scored after three days. The phenotype of clones that scored as negative in the drug replication test was confirmed by a "mass test" (ORIAS and HAMILTON 1979). 0.2 mll of stationary cells was inoculated into 2 0 ml of CHX, MP15 or MP75 medium in small test tubes at room temperature and scored for growth five days later.

Identification of true progeny: True progeny are clones that possess a newly developed macronucleus after conjugation. In many cases, at least one of the parental clones was a functional heterokaryon, phenotypically sensitive to cycloheximide or 6-methylpurine, and true progeny clones could be identified by their newly acquired resistance to these drugs. In rare cases where these markers were absent, true progeny clones were identified by their immaturity, as indi- cated by a mating test (maturity test) described in the next section. (Nonconjugant clones retain the old macronucleus and remain sexually mature; ALLEN 1967; NANNEY 1956 ) Clones were tested against at least two nonparental mating types and were considered immature and retained for further work if they did not form pairs. Immature clones were transferred to maturity and accepted as true progeny by the most stringent criterion of having nonparental mating types.

Mating tests: Mating tests were performed to identify potential true conjugant clones immediately after conjugation, and later to determine the mating type of a clone (by testing it against all mating-type testers). The methods of O R I 4 S and FLACKS (1973) were used with the exception that the two-day-old testers and the clone to be tested were mixed without feeding, and the results were scored six to eight hr later.

Food vacuole formation tests: The ability of a clone to form food vacuoles was determined by ink tests (ORIAS and POLLOCK 1975). Clones were replicated to 0.05 ml of EPP in the wells

Sterile stock cultures and derivatives were grown at room temperature in 100 X 13 mm

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1064 P. B. SUHR-JESSEN A N D E. ORIAS

of UV-sterilized flat-bottomed microtiter plates and incubated for two days at 39". Equal volumes of newly made, prewarmed INK medium were then added and the incubations continued for one hr. The clones were killed with formaldehye (final conc. 2% v/v) and scored for food vacuole formation with an inverted microscope (Leitz Wezlar, 16 x 10 or 40 x 10). While more than 95% of the cells in wild-type clones had formed vacuoles that contained ink, less than 5% of the mutant (vac) cells contained such vacuoles. Intermediate results were occasionally found, in which case the clones were tested again. Clones were considered vac- if in both tests less than 10% of the cells contained ink vacuoles.

Mass selection of progeny

In some crosses where less than 5% of the cells formed pairs, it was possible to select true conjugants by exploiting their resistance to cycloheximide (and the sensitivity of the parental heterokaryons). One ml of mating mixture was transferred to 10 ml of PPF medium at eight h r after the cultures were mixed to allow conjugation. After three days of incubation at 30", an equal volume of cycloheximide medium (final concentration 15 gg per ml) was added. Survivors were cloned, tested for food vacuole formation and transferred by serial replication to maturity.

Isolation of v a c mutants

To isolate mutants, clones CU329 and CU324 were crossed at 30" according to the protocol described above, with two modifications: (1) clone CU324 was mutagenized with N-methy1-N'- nitro-N-nitrosoguanidine as described by ORIAS and FLACKS (1973), and (2) cytogamy was induced as follows (ORIAS, HAMILTON and FLACKS 1979). Four and three-quarter hr after starved cultures were mixed, a sample of the conjugation mixture was diluted into PPF medium (or PPF -l- 10 mM CaCl,), and kept at 30" in this medium for at least 30 min. Pairs were then isolated into PPF medium. After three days at 30", these "drop" cultures were replicated to MP75 medium to eliminate nonconjugant clones.

The 6-methylpurine survivors were then screened for food vacuole formation at 39". v a c candidates were consecutively cloned and tested for food vacuole formation two more times. The frequency of cytogamy was monitored in each experiment by testing a sample of 200 to 300 pairs by serial replications to MP15 and CHX media (in both sequences), as described by ORIAS and HAMILTON (1979).

In early experiments, several tiny and monster vac- clones were seen, but only clones with normal cell morphology at 39" were kept. Later, all vac- cells were accepted, regardless of overall cell morphology.

RESULTS

Isolation of vac- clones Mutants with temperature-sensitive food vacuole formation were sought using

the protocol outlined in Figure 1. Among approximately 40,000 clones screened, 40 clones of independent origin were isolated by the criterion that at least 95% of the cells are unable to form food vacuoles when tested after two days of growth in EPP medium at 39". At 22", all the mutant clones formed food vacuoles at the same rate as the parental wild-type control; at 30" only two (SJ180 and SJISS) failed ot maintain the wild-type rate.

The mutants were all phenotypically sensitive to cycloheximide and resistant to 6-methylpurine (data not shown), as expected if indeed they arose from cytogamy and not cross-fertilization. Six vac- mutants expressed additional defects at the restrictive temperature: cell division (SJ185 and SJ186), ciliary row orientation (SJI 89, SJ191), oral apparatus and contractile pore morphology (SJ188) and ability to increase cell mass (SJ184).

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FOOD VACUOLE-LESS MUTANTS O F TETRAHYMENA 1065

CU329

x PAIR

,-b CG INDUCTION ,-& ISOLATION (100%)

MUTAGEN IZED

CU324

~ R D INK 1sT INK 6np SELECTION

SCREENING 4-8 SCREENING 4-8 (21.7% XF +

(0.U VAC-) (0.6% VAC-) 8,9% CG)

FIGURE 1 .-Protocol for the isolation of micronuclear u a c mutants of Tetrahymena thermo- p h i l ~ ~ . CG and XF mean cytogamy and normal crossfertilization, respectively. The second ink screening has been omitted. The fate of the isolated pairs was as follows: 29% died; 28% were 6-mp-S (mainly nonconjugants and excytogamous progeny derived exclusively from CU329), 8.8% were lost through accident or contamination and the rest were 6mp-R.

Genetic analysis of vac- clones Although the selection procedure allows the isolation of homozygous micro-

nuclear mutations, the defects could instead have been macronuclear, mitochon- drial or cortical. The mutation(s) could be dominant or recessive and within one or many complementation groups. To discriminate between these possibilities, a series of crosses was performed. The results expected on the assumption that the mutants have micronuclear homozygous recessive defects are shown in Figure 2.

The vac- clones were first crossed to CU329 or CU330. All the progeny formed vacuoles at 39” (Table 2 ) . The Fl’s were then backcrossed to their parental mutant clone. The 1:l segregations found in most of the crosses (Table 3) are consistent with the assumption of a micronuclear recessive defect(s) in food vacuole formation. The restrictive temperature for food vacuole formation in SJ190 is close to the incubation temperature of the ink tests; the higher than expected nuniber of vac+ cells is probably due to leakiness of the mutation. Only wild-type progeny were found in backcrosses of SJ183 and SJ187, but the lack of vac- progeny is not considered significant in view of the small number of progeny and the results of F, X F, crosses. SJ189 and SJ191 formed pairs, but were sterile, i.e., produced no true progeny. SJ180 and SJ188 were not crossed to their F,’s due to sterility and/or difficulties in pairing.

Two Fl’s from each mutant were crossed to produce an F2. The 3:l segregations found among most of the crosses confirm that the mutant clones have a micronuclear recessive defect(s) for €ood vacuole formation (Table 4). The number of vac- clones among the F, progeny of SJI 79 is high, and suggests that one of the Fl’s could be homozygous rather than heterozygous for the vac- mutation in its germ line. Similar abnormalities have been detected occasionally (ORIAS and HAMILTON 1979); their cytogenetic basis has not been investigated. Four clones

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1066 P. B. S U H R - J E S S E N A N D E. ORIAS

Fl loo%,= .

9

5 Fl

B

loo%=.- 0

D

vac A

Fl vac- - 0 x 7 , .

C

vac B

E FIGURE 2.-Expected phenotypes and frequencies of true progeny clones in various types of

crosses performed with putatively recessive UUF mutants and their F, progeny. A: mutant crosses to wild type. B: F, x F,. C: F, x mutant (backcross). D: cross of two mutants with different mutations within the same complementation group. E: cro,ss of two mutants with mutations in different complementation groups. x: mutation. Closed square: wild type. Open square: mutant.

TABLE 2

Phenotypes of vac- clones and their F , progeny

Vac- Morphology Total clone OA Cell isolated

SJ140 SJ176 SJ177 SJ179 SJ180 SJ183 SJ184 SJ185 SJ186 SJ187 SJ188 SJ189 SJ190 SJ191

W t wt W t W t Wt Wt Wt Wt W t Wt Wt W t

? Tiny W t Monster Wt Monster W t wt OA- Bloated CVP-

Twisted Twisted w t Wt

Twisted Twisted

1245 384 192 9 w

M t 96t

248 288 192

384 192

Dead

286 189 141 30

18 58 96 92

112

121 64

Pairs

NC

4 9 140 120 64

29 10

119 173 69

119 124

550 Of 55 0 21 0 2 0 28 0 1 0

28 0 33 0 23 0 11 0 38 0 28 0

144. 0 4 0

Results from several crosses were pooled unless otherwise specified. OA: oral apparatus.

* Exconjugants were separately tested in some cases. + One experiment only. 8 Clones obtained after mass selection for F, progeny; the parental clones formed pairs, but

NC: nonconjugants. CVP: contractile vacuole pore. Wt: wild type.

the fertility was well below 1 %.

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FOOD VACUOLE-LESS M U T A N T S OF T E T R A H Y M E N A

TABLE 3

Phenotypes of back-cross progeny of F , clones

1067

Cell Backcross pairs F, from phenotype* of Total Progeny p CXZ)

uac- clone oriKinal mutant isolated Dead NC uac+ uac- (1:i rabo)

SJ140 SJ176 SJ177 SJ179 SJ183 SJ184 SJ185 SJ186 SJ187 SJ189 SJ190

57w 6 72t 288t 96 96

Monster 192 Monster 96

96 Twisted 192

192 192 2881

Tiny 192t

170 21 6 100 70 54

168 81 65 33 98 67

103 108

267 395 101 20 40 17 90 29 61 94 91 71

141

68 71 33 28 25 22 2 4 2 0 3 4$

10 11s 0 2 2 0

25 9 10 8 24 15

_ -

0.2-0.3 0.1-0.2 0.3-0.5

0.7-0.9

0.00 1-0.01 0.5-0.7 0.1-0.2

SJ191 Twisted 196 87 109 - -

For abbreviations see Table 2. * If different from wild type. j- Pooled homogenous data. $ Two of these were also tiny. s Four of these were also monsters.

TABLE 4

Phenotypes of F , progeny of vac- clones

Cell F, from phenotype’ of Total

uac- clone onginal mutant isolated+ Dead

SJ140 288 177 SJ140 480$ 139 SJ140 96$s 25 SJ176 192 34 SJI 76 1922 26 SJ177 192 62 SJ179 192 103 SJ183 96s 74

SJ185 Monster 96s 10 SJ185 Monster 96$$ 22 SJ186 Monster 96s 30 SJ187 192s 100 SJ187 192$s 46 SJ189 Twisted 386 94

SJ184 Tiny 96s 55

Fl X F, pairs Progeny p cxz,

NC uacf uac- (3:l ratio)

156 217 27 27 21

130 55 17 15 19 15 30 39 68

289

9 96 27

103 101

18 4

16 44 46 29 36 57 2

-

6 28 17 28 44

16 1

234j 13 7

17 21 1

-

IO//

0.3-0.5 0.1-0.2

0.0 1-0.05 0.7-0.9 0.1-0.2

0.001-0.01

0.1-0.2 0.05-0.1

0.5-0.7 0.3-0.5 0.24.3 0.2-0.3 ___

SJ190 288 125 65 70 28 0.5-0.7

For abbreviations see Table 2. * If different from wild type. t Pooled homogenous data.

Single experiment. 1 S’ ix were also tiny. ‘1 Thirteen were also monsters.

$ F, progeny.

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1068 P. B. S U H R - J E S S E N A N D E. ORIAS

TABLE 5

Phenotypes of progeny of crosses of vac- clones to SJI40

Pairs Cell Total Progeny

Vac- clone Dhenotwe’ isolatedt Dead NC vac+ vac- - “~

S J I W 288 50 63 0 176 SJ176 SJ179 SJ180 SJ183 SJ184

’ SJ185 SJ186 SJ187 SJ188 SJ189 SJ190 SJ190 SJ191

9 6 9%

9% Tiny 528

Monster 768 Monster 480

288 Bloated OA-, CVP- 480

864

384

Twisted

96s Twisted 96s

42 34 25 71

221 163 78 10

226 158 199 350 249 118 256 32 86 394

590 141 27 38 25 64.

0 20 _ _ _ -

0 8 0 144 0 219 0 113

_ - 411

0 203 31 0 0 7

Defective gene

vacA vacA

vacA vacA vacA vacA

vacA vacA

vacA

For abbreviations see Table 2. * If different from wild type. + Pooled data. $ A vac- F, was crossed to the original mutant.

One experiment. After mass selection of true conjugants.

could not be rigorously analyzed because of sterility. These clones most likely have micronuclear mutations since (a) 1 :I segregations among the backcross progeny of SJ177 were found (Table 3), (b) SJ191 failed to complement with SJ140 (Table 5), and (c) vac- vegetative assortants were obtained from FI’s of SJ180 and SJ188, indicating the transmission of a germ-line mutation to the F1 progeny (see DISCUSSION for a review of phenotypic assortment).

No evidence of linkage of the vac- mutations to either the C h A or Mpr loci was found.

Relationship of additional mutant phenotypes to the vac- mutations As noted above, six vac- mutants exhibited additional defects at tlie restrictive

temperature. Are these other defects determined by the same mutation that deter- mines the uac- phenotype? The “tiny” phenotype of SJI 84 and the “monster” phenotype of SJ185 segregated among F, and backcross progeny as if due to a separate recessive mutation(s) not closely linked to the vac- muta6ons (Tables 3 and 4, respectively). Since all the tiny and monster phenotypes appeared among the vac- progeny, we assume that these two types of cells are pleiotropi- cally unable to form food vacuoles. The F, ratios obtained (16 vac+ : 4 vac- normal : 6 vac-, tiny and 44 vac+ : 10 vac-, normal : 13 vac; monster) are not significantly different from the expected 9:3:4 ratios (probabilities by x2 test: P = 0.8-0.95 and 0.2-0.5, respectively). For mutants SJ186 and SJ189, the additional phenotype was not recovered among the F, and/or backcrosses, but

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since some otherwise definitely normal vac- segregants were obtained, it seems likely that the additional phenotype is also determined by a separate mutation (s) . For SJ189, this conclusion is strengthened by the isolation of a nontwisted vac- vegetative assortment subclone from an initially vac+ F, clone (see DISCUSSION).

Mutant SJ188 behaved differently from the above mutants. At 30°, it has a missing or rudimentary OA (OA-) ; the cell has a bloated appearance that is accompanied by defective contractile vacuole pores (CVP) . Genetic analysis of this clone could not be carried past the F, because of sterility. Nevertheless, vac- phenotypic assortants were obtained among the F, clones. All of these were also bloated, OA- and C V P at 30”. The failure of these traits to assort independently during vegetative growth is consistent with the possibility that the complex phenotype of SJ188 is determined by a single mutation (see DISCUSSION). (A second class of segregants from SJ188 gave rise to clones that at 22” contained about 40% wild-type cells, while the rest were nonvacuole formers, had normal OA and CVP’s, and lacked a micronucleus. These segregants behaved as “semi- amicronucleate clones” described by NANNEY (1957) , with defects probably unrelated to the phenotypes involved here.)

The sterility of SJ191 precludes any comment on the genetic relationship of its two defects.

Complementation tests of vac- mutants To determine the number of complementation groups represented among the

vac- clones, double heterozygotes were constructed and tested for food vacuole formation. The appearance of vacuole formers would indicate complementation (Figure 2, panel E). Results from crosses of SJ140 to the other mutants are shown in Table 5. It is apparent that SJ140, SJ176, SJ183, SJ184, SJ185, SJ186, SJ189, SJ190 and SJ191 all belong to one complementation group, designated vacA. (In one of the crosses between SJ190 and SJ140, double heterozygotes were scored as vac+, but this probably reflects the leakiness of the vac- mutation already discussed, rather than complementation.) Double heterozygotes could not be obtained from SJ177, SJ179, SJ180, SJ187 and SJ188 due to their sterility.

All the uacA mutants behaved functionally as if they had a defect in OA development, but their OA appears normal under optical microscopy ( SUHR- JESSEN and ORIAS, in preparation).

Double heterozygotes were also obtained by crossing Fl’s of various mutants to SJ140. One-half of the progeny should be single heterozygotes and therefore vacuole formers, as shown in Figure 2A. The other half should be vacuole formers only if the mutations complement. The results (Table 6) confirm that SJ140, SJ176, SJ183, SJ184, SJ185, SJ186, SJ189 and SJ190 belong to the same (vacA) complementation group and they suggest that SJ177, SJ179 and SJ187 also belong to this group. Two crosses, involving Fl’s of SJ185 and SJ189, resulted in lower than expected numbers of vac- clones. The low number of vac- clones among progeny with the SJ190 mutation is again most likely due to leakiness.

Attempts to construct double heterozygotes with SJ180 and SJ188 failed; less

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1070 P. B. SUHR-JESSEN AND E. OKIAS

TABLE 6

Phenotypes of progeny of crosses between B,’s of vac- clones and SJI40

F from :lolll3

Cell phenotype‘ of Total

original mutant isolated? Dead

SJ14Q SJ176 SJ177 SJ179 SJ180 SJ183 SJ184 SJ185 SJ185 SJ186 SJ187 SJ188 SJ189 SJ190 SJ190

768 720 672 963

960 192%

Tiny 1 92 Monster 480

Monster 672 192

Bloated OA-, CVP- 960 Twisted 672

480 672

Monster 963

245 286 280 20 54

113 74

215 46

191 I20 73

253 156 243

Pairs

NC

373 272 274 32

906 76 31

152 30

289 31

887 321 288 273

Progeny vac+ uac-

77 73 84 78 69 49 24 20

2 1 47 4.0 58 55 17 4

108 84 24 17

61 37 20 16

127 29

_ -

_ _

p cxz, (1:l ratio)

0.9-0.95 0.7-0.9 0.3-0.5 0.5-0.7

0.1-0.2 0.7-0.9

0.001-0.01 0.7-0.9 0.5-0.7

0.00 1-0.0 1 0.7-0.9 <0.001

For abbreviations see Table 2. * If different from wild type. t Pooled homogenous data. 2 One experiment.

than 1 % of the cells formed pairs, and among these none (less than 0.1 % ) gave rise to true conjugants (Table 6), Mass selection for true progeny was not feasible since we failed to obtain functional heterokaryons for cycloheximide or 6-methylpurine resistance from these clones (data not shown). SJ180 appears to be defective in phagocytosis per se and SJ188 has-if at all-only a rudimentary OA (SUHR-JESSEN and ORIAS, in preparation). Since both of these clones differ phenotypically Gom the uacA mutants, it would not be surprising if they belonged to two complementation groups other than uacA.

Two double heterozygotes, SJ14O/SJ176 and SJ14O/SJ179, were used in an attempt to detect wild-type phenotypic assortants. Such assortants might have been expected if the two mutations were in different DNA base pairs and if intracistronic recombination could occur in the macronucleus. No wild-type assortants were found, even though the heterozygotes were grown for more than 500 doublings in EPP medium at the restrictive temperature. This lack of phenotypic assortment supports the idea that the mutations, if they affect different base pairs, are closely linked, as expected from their lack of complementation.

In addition to the 12 uac- mutant clones of independent origin, 26 other clones have been assigned to the uacA complementation group by crosses to each other and to SJ140. These 26 clones were isolated together with SJ14Q in an experiment in which the mutagenized cells were allowed to undergo six doublings prior to the induction of self-fertilization, and resulted from screening 5,000 clones out of a mutagenized sample of about 500,000 pairs. It is likely (but not certain)

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FOOD VACUOLE-LESS M U T A N T S O F TETRAHYMENA 1071

that most of these clones are of independent origin. The combined results suggest that, with the methods used in this study, temperature-sensitive defects were recovered up to an order of magnitude more frequently within the uacA gene than within other genes presumably also required for food vacuole formation.

Mutant SJ138 is a heterokaryon Clone SJ138 was isolated in the same way as the other mutants and is also

phenotypically vac-, 6-mp-R and cycl-S. When crossed to wild-type clone BIII, all true F, progeny (58 of the 96 clones isolated) were vac+, 6-mp-S and cycl-R rather than vac+, 6-mp-R and cycl-S as expected. All of the 290 F, progeny tested were vac+ and 6-mp-S; 211 of them (or 72.8%) were cycl-R. These results demonstrate that SJ138 is homozygous in its micronucleus for the alleles deter- mining vac+, cycl-R and 6-mp-S. Thus, the micronucleus has markers derived exclusively from one parent (CU329), while the macronucleus has markers derived exclusively from the other (CU324). This unexpected genetic compo- sition is further considered below.

DISCUSSION

Use of cytogamy to isolate mutants Cytogamy is a form of conjugation, first described in Paramecium by SONNE-

BORN (1975), where the migratory gametic nuclei are not exchanged between mates; instead, they fuse to their sister stationary gametic nucleus and then continue with the normal process of mitosis and differentiation into new macro- and micronuclei. Since the two gametic nuclei are mitotic daughters of a single functional haploid product of meiosis, cytogamy makes each exconjugant an “instant” whole-genome homozygote. An inducible process with identical genetic consequences has recently been discovered in Tetrahymena ( ORIAS, HAMILTON

and FLACKS 1979; ORIAS and HAMILTON 1979), and was used in this study. The protocol used here to isolate the recessive vac- mutants exploited the induc-

tion of cytogamy with 2% proteose peptone, believed to act through hyperosmotic shock (ORIAS, HAMILTON and FLACKS 1979). On the average, cytogamy was induced in 9% o€ the treated pairs (Figure 1) , about one-third of the frequency reported for nonmutagenized cells ( ORIAS, HAMILTON and FLACKS 1979). The difference could be due to the presence in the micronucleus of induced lethal mutations, alone or combined with a possible slight change in the optimal time for shock treatment or decrease in conjugation synchrony, both of which could have been induced by the mutagenic treatment. Slight differences in optimal induction time are known to exist among different T. thermophila strains (ORIAS and HAMILTON, personal communication). Nevertheless, the method is still about 20 times more efficient at generating whole-genome homozygotes than short- circuit genomic exclusion (BRUNS, BRUSSARD and KAVKA 1976), and requires a less tedious protocol than genomic exclusion (ALLEN 1967; ORIAS and FLACKS 1973).

The use of a recently isolated mutation conferring resistance to 2-deoxygalac-

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1072 P. B. SUHR-JESSEN AND E. ORIAS

tose which, under certain conditions, behaves as recessive (ROBERTS and ORIAS, unpublished observation) , permits selection of excytogamous progeny and should eliminate the need to isolate single pairs by hand. Instead, the osmotically shocked conjugating mixture is distributed to microtiter plates with a multichannel dis- penser, so that each well gets on the average one excytogamous pair (ORIAS and FLACKS, unpubiished observation) , following an approach to mutant cloning already used successfully with short-circuit exclusion (ORIAS and BRUNS 1976; BRUNS and SANFORD 1978).

In order to be able to select (rather than screen for) the vac- mutants, attempts were made to kill vac+ cells selectively with agents that could have been expected to require a functional OA for their uptake. Amygdalin (laetrile) and highly potent preparations of diphtheria and ricin toxins were tested without observing significant differences between vac+ and vac- cells. In fact, Tetrohymena cells turned out to be highly resistant to these compounds, whose activity was confirmed by treating sensitive mouse L cells.

About 60% of the vac- isolates were sterile, in the sense that they either failed to form pairs or to produce viable progeny that developed a new macronucleus when crossed at permissive temperatures. This frequency is higher than reported for excytogamous progeny of the same clones when not mutagenized (ORIAS and HAMILTON 1979). Furthermore, six NG-induced 2-deoxygalactose and 2-deoxyglucose-resistant mutants isolated after a similar treatment were all fertile (ROBERTS, personal communication). The higher incidence of sterility among vac- mutants could be due to separate mutations in genes required for the normal execution of the conjugation program, or to secondary consequences of the defect in the OA itself, if these are expressed at a lower restrictive temperature than the capacity to form food vacuoles. The fertile clones have remained so, some for more than 1,000 cell generations.

A macronuclear vac- mutant The vac- phenotype of SJ138 was shown to be due to a mutation(s) present

exclusively in the macronucleus (see RESULTS). This mutant turned out to be a heterokaryon for ChxA and Mpr markers, whose macronucleus is derived exclusively from one parent and its micronucleus exclusively from the other. Conse- quently, we suggest that this clone arose from a normally initiated conjugation in which gametic nuclei were exchanged, but failed to fuse. The stationary nucleus and thc received migratory nucleus (or their mitotic daughters) subse- quently developed independently into the new macro- and micronucleus, respectively. Autoradiographic evidence for a failure of the gametic nuclei to fuse during conjugation in T. thermophila has recently been obtained (HAMILTON and SUHR-JESSEN, in preparation), as well as genetic evidence that hyperosmotic shock also induces fusion failure, but at a lower frequency than cytogamy (HAMILTON, ORIAS and SUHR-JESSEN, unpublished).

Alternatively, SJ138 could be a result of somatic crossing over after the formation of the zygotic nucleus and prior to the first post-zygotic nuclear division. Somatic crossing over has not been demonstrated in T . thermophila at any stage,

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but its possible occcurrrence in the mature macronucleus has been previously suggested (ORIAS 1973). Wild-type phenotypic assortants that could have been formed by somatic crossing over have been sought but not found among double heterozygotes for closely linked mutations (FRANKEL et al. 1976; BLEYMAN and BRUNS 1977; see RESULTS). In none of these three cases, however, was there rigorous independent evidence (obtainable from meiotic recombination) that the mutations indeed were in different base pairs. An explanation for SJ138 based on somatic crossing over must also include the constraint that the three loci (vac, ChzA and Mpr) are located on the same chromosome, all distal to the putative crossover point with respect to the kinetochore. This mechanism requires more ad hoc assumptions and thus seems less probable than the fusion failure hypothesis.

The first v a c clone isolated, NPI, behaves like SJ138 in having its vac- muta- tion restricted to the macronucleus (SILBERSTEIN, ORIAS and POLLOCK 1975). Because NP1 's parents were not genetically marked, the possibility cannot be excluded that it arose directly as a heterokaryon by the same mechanism as SJ138. An explanation based on fusion failure or early somatic crossing over would indeed seem to be more satisfactory than the ones suggested in SILBER- STEIN, ORIAS and POLLOCK (1975) , because a homogeneous mutant macronucleus is generated immediately in a single step.

The number of vac genes We have genetically analyzed nearly 40 fertile vac- clones. Fourteen of these

are of independent origin, having been obtained in separate mutagenesis experiments, and 26 others were isolated in one experiment where six doublings occurred between mutagenesis and conjugation. The statistics (see RESULTS) make it very likely (but not certain) that most of these mutants were also of independent origin. All but two (SJ184, SJl85) of all these clones behave as if they have a single recessive mutation. All but two of the mutations (SJ180, SJl88) belong to a single complementation group, the vacA gene.

The OA is a complex organelle, undoubtedly requiring the function of many genes for its development and function. I t is puzzling that mutations at the uacA gene were recovered with up to an order of magnitude higher frequency than mutations in all other putative vac genes combined. We don't readily see how our protocol for mutagenesis and mutant isolation could so severely restrict the kind of vacuole-less mutants obtained. Since the potential temperatwre-sensitive mutants were always propagated and conjugated at permissive temperatures, any lethal pleiotropic effects of the mutations should not bias the mutant sample recovered. Nor does it seem likely that some ill-understood defect of our complementation tests resulted in spuriously negative results; the absence of phenotype assortment seen in two double heterozygotes under conditions OI selective pres- sure for the wild-type assortments is consistent with the idea that the two mutations in each case are closely linked and perhaps in the same gene. Further- more, analogous complementation tests based on the phenotype of double heterozygotes have been successfully used in the case of the cell division mutants of

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1074 P. B. SUHR-JESSEN A N D E. ORIAS

T. thermophila studied by FRANKEL et al. (1976). Some alternative explanations that cannot be excluded are: (1) the v m A gene is a “hot spot” for mutagenesis, either because of a high probability of mutagenesis per base pair or because it is a very long gene; (2) most of the genes required for oral apparatus development and for phagocytosis are located in a single operon, and all the mutations obtained are severely polar, (3) the haploid genome contains more than one copy of most of the vac genes, while the uacA is a single-copy gene. Explanation (2) would be unprecedented among eukaryotes.

Pleiotropic vac- mutations Six of the vac- mutants exhibit additional defects at the restrictive temperature.

It is important to distinguish whether or not those defects are determined by the same mutation that causes the vac- phenotype. We have used two approaches. The first is the universally applicable method of looking for vac- F, segregants that lack the additional defects. The second approach, unique to Tetrahymena, is based on macronuclear phenotypic assortment (SONNENBORN 1975), a phenomenon in which heterozygous (e.g., F,) clones vegetatively generate subclones that are hereditarily fixed for the expression of only one (either one) of the alternative alleles. This fixation is now believed to be due to a somatic genetic drift caused by the random distribution of the multiple allele copies during macronuclear division (ORIAS and FLACKS 1975; NANNEY and PREPARATA 1979). Different loci Vegetatively assort independently of each other, even if they behave as linked during meiotic transmission (ALLEN 1965; DOERDER 1973), a phenomenon that has been termed dislinkage (WILLIAMS et al. 1978). The basis for macronuclear dislinkage is not known, but macronuclear crossing over and chromosome fragmentation are considered likely alternatives (ALLEN and GIB- SON 1974; RAIKOV 1976; DOERDCR, LIEF and DEBAULT 1977; WILLIAMS et al. 1979). Whichever mechanism is correct, the appearance of vegetative vac- segre- gants (from phenotypically vac+ heterozygous F, clones) that lack additional defects is taken to mean that separate mutations, almost certainly located in different genes, determine the multiple phenotypes.

Three cases remain where pleiotropic effects may be caused by a single mutation. The first example is vac- mutant SJ188, which has a bloated appearance, and shows defects in OA morphology and contractile vacuole pores (as determined by silver impregnation and optical microscopy) at restrictive temperatures. Although sterility prevented a complete geretic analysis, the determinant for the additional defects co-assorted with the vac- phenotype among F, clones. Although this coassortment cannot be rigorously iriterpreted in view of the small sample involved and uncertainty- concerning the mechanism of dislinkage, it is at least consistent with the possibility that the whole phenotypic syndrome is determined by a single mutation.

For two mutants, it was possible to distinguish two mutations; a uacA mutation and a separate, unlinked mutation (or mutation cluster) conferring heat sensitivity for cell growth (SJ184) and cell division (SJ185). It is interesting that the 9:3:4 F, ratios obtained (Table 4) suggest that the second mutation also conferred

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a vac- phenotype. In view of the ubiquity of some of the OA protein components in the cell, it is not surprising to find pleiotropic effects of uac- mutations. The backcross ratio for SJ185 (Table 3 ) , however, does not readily confirm this interpretation.

The present study demonstrates the feasibility of obtaining gem-line mutants of T. thermophila that are defective in oral development; provided that ways can be found to increase the efficiency of recovery of mutations in genes other than vacA, such mutants should make possible the genetic dissection of organelle morphogenesis in a eukaryotic cell, as well as a further elucidation of the role of the food vacuole in nutrient uptake (RASMUSSEN 1976; ORIAS and RASMUSSEN 1979; SUHR-JESSEN and ORIAS, in preparation).

We thank JOHN COLLIER for his gift of diphtheria toxin, STEPHEN BENSON for suggestions for preparing ricin toxin extracts, DONNA WORTMAN for help with control tests of diphtheria and ricin toxin in mouse L cells, MIRIAM FLACKS for providing the mutagenized cultures, and CHARLES ROBERTS, JR., EILEEN HAMILTON and MIRIAM FLACKS for critical comments on the manuscript. Support by The Danish Natural Science Research Council and The Carlsberg Foundation to P. B. SUHR-JESSEN and Public Health Service Grant GM-21067 to E. ORIAS is gratefully acknowledged.

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