Copyright 0 1988 by the Genetics Society of America

Pronuclear Fusion Failure: An Alternate Conjugational Pathway in Tetrahymena themophila, Induced by Vinblastine

Eileen P. Hamilton,* Peter B. Suhr-Jessen? and Eduardo Orias* *Department of Biological Sciences, University of California, Santa Barbara, California 93106, and ?Department of Anatomy, Odense

University, Campzrsvej 55, DK-5230 Odense M , Denmark Manuscript received September 9, 1985 Revised copy accepted January 6, 1988

ABSTRACT Vinblastine is shown to induce pronuclear fusion failure in conjugating Tetrahymena thermophila.

In this alternate conjugational pathway gametic pronuclei are exchanged between conjugants but do not fuse. Each pronucleus undergoes one mitotic division to produce a new macro- and micronucleus. Genetic consequences of pronuclear fusion failure include the following: (1) the progeny are whole genome homozygotes with nuclei derived from single meiotic products, and (2) half of the progeny are heterokaryons with micro- and macronuclei of different genetic origins. These facts make this process extremely useful in strain construction and mutant isolation. The induction of pronuclear fusion failure by vinblastine suggests that microtubules play an essential role in pronuclear fusion.

G AMETOGENESIS and fertilization have well known genetic consequences but their under-

lying mechanisms are incompletely understood (Go- LUBOVSKAYA 1979; SCHATTEN 1982, 1984; SZABO and O'DAY 1983; WASSARMAN 1987). Much can be learned by examining these processes in unicellular eukary- otes which can easily be manipulated both genetically and biochemically.

Tetrahymena thermophila is a single celled organism which by virtue of its extensive biochemical (HILL 1971) and genetic (ELLIOT 1973; SONNEBORN 1974) characterization and tractability, is a suitable model system for the study of gametogenesis and fertiliza- tion in eukaryotes. As a ciliate, it contains two types of nuclei, a diploid (2N = 10) micronucleus and a 45-ploid macronucleus (GOROVSKY 1973). Micronu- clear genes are not expressed during vegetative growth (MAYO and ORIAS 1981). The only known role of the micronucleus is that of a germline nucleus, i e . , a repository of the genetic information for the nuclei formed during sexual reorganization (conju- gation). By contrast, the macronuclear information is actively transcribed and translated during vegeta- tive growth; it determines the phenotype of the cell.

Sex in Tetrahymena involves the partial fusion of two cells of differing mating types, meiosis of their micronuclei, reciprocal exchange and fusion of ga- metic pronuclei (cross-fertilization), and differentia- tion of new micro- and macronuclei from the zygotic nucleus (NANNEY 1953; R A Y 1956; SONNEBORN 1974). Failures in this developmental pathway can be in- duced by a variety of nonlethal treatments. For example, the reciprocal transfer of gametic pronuclei can be blocked by hyperosmotic shock (ORIAS, HAM-

Genetics 118: 627-636 (April, 1988).

ILTON and FLACKS 1979; ORIAS and HAMILTON, 1979) or with a pulse of a microtubule disrupting drug (HAMILTON and SUHR-JESSEN 1980); this block results in self-fertilization (cytogamy).

In this paper we present autoradiographic and genetic evidence that vinblastine induces pronuclear fusion failure in T. themnophila. Pronuclear fusion failure occurs when the resident stationary pronu- cleus and the newly exchanged migratory pronucleus do not fuse. Instead of the fertilization nucleus dividing to produce the new nuclei, each gametic pronucleus gives rise to a macro- and micronucleus. We show that fusion failure generates heterokaryons with a diploid micronucleus derived from a meiotic product from one parent and a macronucleus derived from a meiotic product of the other parent. The significance and the practical utility of these results are discussed.

MATERIALS AND METHODS Strains: Four derivatives of inbred strain B of T. ther-

mophila were used (Table 1). All were functional hetero- karyons for two unlinked markers.

Media: Cells were grown in 2% proteose peptone broth (PP) to which 10 PM FeCls and 250 pg/ml each of penicillin G and streptomycin sulfate were added (ORIAS and HAM- ILTON 1979). For drug resistance tests this medium was supplemented with: 15 pg/ml cycloheximide (CHX), 15 Kg/ ml 6-methylpurine (6MP), or 0.25% 2-deoxygalactose (DGAL). Cells were starved in DRYL'S (1959) phosphate buffer prior to mating.

Induction of fusion failure: Cultures of SB104 and SB105 (Table 1) were grown separately as previously described (ORIAS and HAMILTON 1979). Each was washed into DRYL'S inorganic buffer (at a final concentration of 2 x lo5 cells/ml and starved overnight at 30". The next

628 E. P. Hamilton, P. B. Suhr-Jessen and E. Orias

TABLE 1

Genotypes and phenotypes of strains used

Strain Micronuclear genotype Macronuclear phenotype Mating type

SB 104 MprlMpr, cdaC2IcdaCZ Sensitive to 6-methyl purine, IV wild type for cell division at 38"

SB 105

SB213

ChXAZiChXAZ, CdaAllCdaAl Sensitive to cycloheximide, wild type for cell division at 38"

galAllgalA1, cdaAlIcdaA1 Sensitive to 2-deoxygalactose, wild type for cell division at 38"

I1

VI

SB2 14 galAllgalA1, cdaCZ/cdaC2 Sensitive to 2-deoxygalactose, VI1 wild type for cell division at 38"

Chx (ROBERTS and ORIAS 1973; BYRNE, BRUNS and BRUSSARD 1978) and Mpr (BYRNE, BRUNS and BRUSSARD 1978) are dominant mutations conferring resistance to cycloheximide (15 pglml) and 6-methyl purine (15 pglml), respectively. The recessive cell division arrest mutations, cdaAl and cdaC2 (formerly mo-1" and mo-3*, respectively), cause temperature sensitive blocks in cell division (FRANKEL, NELSEN andJENK1xs 1977). Expression of the mutant phenotype, at 38" but not at 30", results in large misshapen (monster) cells of type A or C, respectively. galAl was used as a dominant mutation: homozygous and heterozygous progeny carrying this mutation are resistant to 0.25% 2- deoxygalactose (ROBERTS and MORSE 1980) when treated at least 13 fissions after conjugation. gal, Chx, Mpr, c d d l and cdaC2 map in chromosomes 1 , l R , ZR, 4 and 5 , respectively (BRUNS 1982). gal and Chx segregate independently at meiosis.

TABLE 2

Phenotypes expected for testcross progeny of clones derived from the SB104 X SB105 cross according to various conjugation pathways

Phenotypes of testcross progeny

Clone under test SB213 SB2 14

Pathwaf Genotype CHX 6MP CK 38C CHX 6MP CK 38C

XF abcd

SF ab

SF cd

FF a

FF b

FF C

FF d

ChxlChx+, MprIMpr+ R R Normal R R Normal CdaAlCdaA + , cdac/cdac+

ChxlChx, Mpr+lMpr+ R S A monst. R S Normal cdaAlcdd, cdaC+lcdaC+

Chx + lChx+, MprlMpr s R Normal S R C monst. cdaA + IcdaA + , cdaClcdaC

ChxlChx, Mpr+lMpr+ R S A monst. R S Normal cdaAlcdaA, cdaC+lcdaC+

ChxlChx, Mpr + lMpr+ R S A monst. R S Normal cdaAlcdaA, cdaC+lcdaC+

Chx+lChx+, MprlMpr S R Normal S R C monst. cdaA + l c d d + , cdaClcdaC

Chx + iChx+, MprlMpr S R Normal S R C monst. cdaA +lcdaA + , cdaClcdaC

a a and b represent one pair of sister caryonides; c and d represent the other pair of sister caryonides. XF, cross-fertilization; SF, self-fertilization; FF, fusion failure; R, resistant; S, sensitive; CK, cytokinesis; A and C monst., monsters

Under FF, maximum caryonidal diversity is obtained if unlike new micronuclei survive in the two exconjugants. Otherwise, two identical showing the terminal phenotype characteristic of cdaAl and cdaC3 homozygotes, respectively.

pairs of sister caryonides will be obtained (2 X ab or 2 x cd).

morning the cultures were mixed at 30" to start conjugation. (SB104 X SB105) were isolated into drops of PP medium. At various times after mixing (usually 5 hr) the mating After separation, the exconjugants were isolated into sep- pairs were treated with 5 PM (or in one case 3 PM) vinblastine arate drops. Drop cultures were incubated three days at sulfate to induce conjugational abnormalities. After 0.5 hr 30" then replicated to PP, CHX and 6MP media (ORIAS the cells were diluted 20-fold with water to reduce the and BRUNS 1975). The PP cultures were incubated one day concentration of the drug. at 38°C then scored for their cell division phenotypes. After

Phenotypic tests: Single conjugating pairs from the cross a 3-day incubation at SO", the antibiotic-treated cultures

Pronuclear

were scored for drug resistance; survivors were replicated to PP at 38" and to the alternate drug (ie., CHX survivors to 6MP and vice versa).

Test crosses: To determine the micronuclear genotypes of our presumptive fusion failure progeny each fusion failure caryonide was separately crossed to the tester strains SB2 13 and SB2 14 (Table l), 2-deoxygalactose-sensitive heterokaryons. None of the clones to be tested carried the galA allele, so true progeny of the test crosses were selected by their DGAL resistance. SB213 and SB214 are homozy- gous for the recessive wild-type alleles at the ChxA and Mpr loci and homozygous for one of the unlinked, recessive cell division arrest markers (cduAI or cduC2). This allowed us to determine which alleles were present at these loci in the micronucleus of each caryonide.

Conditions favoring cross-fertilization were used (ORIAS and HAMILTON 1979) in all test crosses. Pairs were isolated 7 hr after conjugation was initiated by mixing. The drop cultures were incubated 3 days at 30" then replicated to DGAL medium. Only true conjugants, with copies of galAI in their macronucleus, survive. Survivors were replicated to CHX, 6MP, and PP (38"), as already described (ORIAS and HAMILTOS 1979), to determine whether or not the testcross progeny inherited at least one copy of the domi- nant alleles at the four loci under question (ChxA, Mpr, cdaA and cdaC, respectively). The phenotypes expected for the testcross progeny depending on the conjugation path- way followed by a given SB 104 X SB 105 pair is shown in Table 2.

Autoradiographic assay: All procedures were identical to those described previously (HAMIL.I.OS and SUHR-JESSES 1980). One parental strain was prelabeled with ['Hlthymidine and mated to an unlabeled strain. Self- fertilization was induced by 0.5-hr treatment with vinblas- tine (3 KM) 5 hr after the initiation of conjugation. Late in conjugation (8-10 hr) pairs were fixed onto slides and coated with film. After 1-week exposure the autoradi- ograms were developed and stained with Giemsa.

Reagents: Chemicals were obtained from the following sources: proteose peptone from Difco; penicillin G, strep- tomycin sulfate, cycloheximide, 6-methvlpurine, 2-deoxy- D-galactose and vinblastine sulfate from Sigma. Vinblastine sulfate (10 mg/ml) was dissolved in water and stored at - 18"; other drugs were prepared and stored as previously described (ORIAS and HAMILTON 1979).

RESULTS

Autoradiographic identification of pronuclear fu- sion failure: In previously reported experiments we induced self-fertilization with the antimicrotubule drug vinblastine (HAMILTOS and SUHR-JESSES 1980). The nuclei of one parental strain were labeled with ['Hlthymidine prior to conjugation. When these cells were mated to an unlabeled strain, then treated with vinblastine during conjugation, pairs which self-fer- tilized were easily identified; the unlabeled conjugant (marked by its unlabeled parental macronucleus) contained only unlabeled new nuclei, while the la- beled conjugant (labeled parental macronucleus) had only labeled new nuclei. Cross-fertilization pairs were also seen; in these pairs both conjugants contained labeled new nuclei (Figure 1A).

In addition to these expected types of pairs, an- other kind of pair, with a distinctive labeling pattern,

Fusion Failure 629

e

FIGURE I.-Autoradiograms of pairs after cross-fertilization and pronuclear fusion failure. A, Autoradiogram of a pair in which cross-fertilization occurred. Each cell contains two labeled new micronuclei (m) and new macronuclei (M). These nuclei are division products of the fertilization nucleus. The nucleus at the bottom of each conjugant is the old macronucleus (0); since this nucleus is labeled in the cell on the right this cell is the one prelabeled with ['Hlthymidine. B, Autoradiogram of a pair in which pronuclear fusion failure occurred. Each conjugant contains one labeled and one unlabeled new micronucleus (m) and new macronucleus (M). Each set (labeled and unlabeled) of new nuclei is derived from a gametic pronucleus.

was observed. Each conjugant contained one labeled new macro- and micronucleus and one unlabeled new macro- and micronucleus (Figure 1B). This pattern indicates that the four new nuclei in each cell cannot be derived from a single fertilization nucleus, as is the case with normal cross-fertilizers. Also, although the labeled nuclei could have incorporated DNA from both parents, the unlabeled nuclei must be of uniparental origin. Thus, the simplest expla- nation is that these nuclei originated from unfused (but exchanged) gametic pronuclei: according to the

630 E. P. Hamilton, P. B. Suhr-Jessen and E. Orias

@ ’ k ’ 1

3 &

& 5 k

3.1

4’ .1

5’ J

0 0

9’A

a b C d

scheme shown in Figure 2. This discovery led us to develop a genetic assay which could distinguish be- tween pairs which underwent this new conjugational pathway and the other known pathways (ie., cross- fertilization, self-fertilization and macronuclear re- tention). This allowed us to explore the induction and genetic consequences of pronuclear fusion failure.

Genetic identification of pronuclear fusion fail- ure: Strains SB 104 and SB 105 were used in these experiments since their fusion failure progeny could easily be identified by the production of sister car- yonides with different and complementary drug re- sistance phenotypes (Figure 2). Fusion failure caryon- ide clones can be resolved and identified by their unique pattern of drug resistance when exposed to the sequence of drug selections shown in Table 3. This protocol also allows the identification of other

FIGURE 2.-A diagramatic representation of normal conjuga- tion and pronuclear fusion failure in T. themophila. l , The first visible stage of conjugation occurs when two cells of different mating type fuse anteriorly to form a pair. The micronucleus of each cell undergoes meiosis. 2, Three meiotic products migrate to the posterior of each conjugant and are destroyed. The remaining product divides mitotically producing two identical gametic pron- uclei. 3, Migratory gametic pronuclei are exchanged between conjugants. (This step is blocked in self-fertilization.) 4, The pronuclei fuse. 4’, The pronuclei in each cell do not fuse. 5 , The first postzygotic division. 5 ’ , The first postzygotic division does not occur. 6, The second postzygotic division. The spindles are oriented along the long axis of each cell. 6’, The pronuclei undergo a division analogous to the second postzygotic division. 7 and 7 ‘ , The anterior division products differentiate into macronuclei while the posteriorly located products become the new micronuclei. The old macronucleus moves to the posterior of each cell where it disintegrates. 8 and 8 ’ , The conjugants separate. One randomly selected new micronucleus is destroyed in each exconjugant; the other divides mitotically. 9 and 9’, Products of the first vegetative cell division (caryonides) receive one new macro- and micronucleus. Note that the caryonides produced after normal conjugation are heterozygous homokaryons (9) while of those produced after pronuclear fusion failure two are homozygous homokaryons and two are homozygous heterokaryons (9’). Various homokaryon heterokaryon combinations are possible depending on which new micronuclei are destroyed. Each possible combination is shown in Figure 3 (first column, top three lines). Key to symbols: white and black nuclei represent homozygosity for a recessive or dominant allele, respectively. Half black, half white nuclei are heterozygous.

pathways a pair could follow: cross-fertilization, self- fertilization, or macronuclear retention.

Induction of pronuclear fusion failure: To induce fusion failure, conjugating cells were treated with a 0.5-hr pulse of 5 PM vinblastine beginning 5 hr after mating was started. Among the progeny of different experiments we found that 5-10% of the pairs yielded presumptive caryonides with phenotype com- binations consistent with the predicted scheme for fusion failure (Figure 2). Although all four presump- tive caryonides were not always recovered, 3-caryon- ide and certain 2-caryonide sets could still be iden- tified as having resulted from fusion failure. The rest of the progeny from our various experiments included 20-5596 cross-fertilizations, 10-15% self- fertilizations and the remainder which underwent macronuclear retention or died. The frequency of fusion failures is probably underestimated since the

Pronuclear Fusion Failure

TABLE 3

Phenotypic identification of conjugation pathway

63 1

Results of first replication to: Results of second replication:

Conjugational from CHX to: from 6MP to:

!%::3 Exconjugant clones CHX 6MP PP (38") PP (38") 6MP PP (38") CHX

XF a R R + + R + R + R b R R + + R

SF a R S A A S 0 0 S b S R C 0 0 C

FF a R R A&C A S c S S

0 b S S + 0 0 0 0

b R R A&C A S C

MR a S S + 0 0 0

Progeny exconjugant clones (a and b) derived from individual pairs from a SB 104 X SB 105 cross were replicated as shown to determine which conjugation pathway the pair had followed. Both caryonides of an exconjugant are assumed to survive.

Key to symbols: XF, cross-fertilization; SF, self-fertilization; FF, pronuclear fusion failure; MR, macronuclear retention; a, cytoplasmic derivative of SB105; b, cytoplasmic derivative of SB104; R, resistance to drug; S, sensitivity to drug; +, wild type with respect to cell division at 38"; A, cdaA-determined "monster" phenotype at 38"; C, cduC-determined "monster" phenotype at 38"; 0, no survivors to test after the first replication. For further information see MATERIALS AND METHODS.

TABLE 4

Testcrosses of the four caryonides from presumptive fusion failure pair ~-

Crosses to gal-llgal-1, cdaA-lIcdaA1 tester ~~~ ~

Caryonides Progeny phenotypes

Phenotype CHX-R CHX-S GMP-S GMP-R Total pairs Fertility

Clone CHX 6MP Mon Mon A WT 38" SF NC X isolated (rc)

1 R S A 25 14 9 48 32 2 S R C 12 12 24 48 25 3 R S A 16 29 3 48 33 4 S R C 16 2 9 21 48 38

Crosses to gal-llgal-I, cdaC2lcdaC2 tester

Caryonides Progeny phenotypes

Phenotype CHX-R GMP-S

CHX-S GMP-R Total pairs Fertility

Clone CHX 6MP Mon W T 38" Mon C SF NC X isolated (rc)

1 R S A 32 13 3 48 67 2 S R C 26 2 7 13 48 58 3 R S A 14 34 48 29 4 S R C 29 7 12 48 60

Caryonide clones were crossed to the tester strains and progeny phenotypes determined as described in Materials and Methods. Clones 1 and 2 are sister caryonides, i .e., the two daughter cells of one exconjugant; while clones 3 and 4 are sister caryonides of the other exconjugant cell.

Key to symbols: CHX, cycloheximide; 6MP, 6-methyl purine; R, resistance; S, sensitivity; Mon, monster phenotype ( c d a mutation); A, monster type A at 38"; C, monster type C at 38"; WT 38", wild type at 38"; SF, self-fertilization; NC, nonconjugant; X, dead. Percent fertility was calculated as the number of pairs generating progeny with a new macronucleus divided by the number of pairs isolated.

death of the appropriate two caryonides (a and c, Figure 2) converts the fusion failure pattern into a self-fertilization pattern.

Pronuclear fusion failure produces homo- and heterokaryons: Previous cytological studies (RAY 1956; NANNEY 1953) had led to the conclusion that only one of the two new micronuclei in each excon- jugant remains functional. The two sister caryonides

receive mitotic copies of this micronucleus. Since putative fusion failure exconjugants produce sister caryonides with genetically different macronuclei (Figure 2), we predicted that one of these sister cells should be a heterokaryon and the other a homokar- yon. To test this idea, caryonides from all presump- tive fusion failure pairs were testcrossed to determine their micronuclear genotypes. Table 4 shows the

632 E. P. Hamilton, P. B. Suhr-Jessen and E. Orias

Fusion Failure Caryonides

9 6 9 6 0 6 06 0 6 0 6 Ob 0 0 6 6 0 6 i 0 6 6

f of event

1

2

1

1

1

1

1

1

vg. k t . 4

70

50 60

60

65

25

60

35

I

Furion Failure Caryonides

3 6 6 06 6 9 6 0 6 06 0 0 6 6

0 0 i

Y of event!

1

2

2

1

1

1

1

1

1

IV9. fert. 96

60

40 50 45 40

60

50

50

5

30

I

FIGURE 3.-Summary of fusion failure caryonide genotypes. The genotypes and phenotypes of all our pronuclear fusion failure caryonides. The small circles represent micronuclei (genotypes) while the large circles depict macronuclei (phenotypes). Black circles and white circles indicate homozygosity for the four genetic markers derived from SB104 and SB105, respectively. Half black, half white circles represent quadruply heterozygous nuclei. In these cases we do not know if both caryonides survived as they are identical, hence the central location of these nuclei. An X replacing the micronucleus signifies that the caryonide was infertile and its genotype could not be determined. Missing caryonides denote the death of the clone after conjugation. Sister caryonides, i . e . , derived from the same exconjugant, are pictured closely adjacent to each other. The # of events refers to the number of times each caryonide pattern was observed. Fertility is as in Table 3, averaged over each set of caryonides.

results of a typical set of test crosses of four caryonides from a presumptive fusion failure pair. These test crosses showed that clones 1 and 2 (which were sister caryonides) had the same micronuclear genotype: ChxAZlChxA2, Mpr + lMpr + , cduAl lcduAl , cduC2 + I cduC2 + , although they had different macronuclear phenotypes. The micronucleus of each clone was derived from the gametic pronucleus of only one parent (SB105). Sister caryonide clones 3 and 4 had another micronuclear genotype: ChxA2+lChxA2 +, MprlMpr, cduAl +lcdaAl+, cduCZIcduC2, also of single parent origin (SB104). A summary of the results of similar test crosses, conducted on all caryonide sets identified as fusion failure progeny from their pheno- types, is shown in Figure 3. Some of the caryonides (9 out of 56) were not fertile enough to produce test cross progeny.

All of our test cross results (Figure 3) and less detailed test crosses of 260 additional fusion failure clones (results not shown) supported the predictions of the fusion failure scheme: (a) Each exconjugant generates one homokaryon and one heterokaryon. (b) Every new nucleus (micro- or macro-) has uni- parental genetic origin. (c) The two parental geno- types appear with equal frequency (14: 13) among the micronuclei of the fusion failure progeny. (d)

Sister caryonides, while unlike in macronuclear ge- notype (and phenotype), are always alike in micro- nuclear genotype. These observations support the occurrence of pronuclear fusion failure and confirm the cytologically observed random destruction of one new micronucleus in each exconjugant (RAY 1956; NANNEY 1953). The relatively high fertility of most fusion failure progeny (Figure 3) strongly suggests that the new micronuclei generated are usually (if not always) diploid and totally normal.

The results in Figure 3 also show that fusion failure is generally bilateral, i.e., if it happens in one conju- gant, it generally happens in the other. In three pairs, however, pronuclear fusion occurred in the mate of a fusion failure conjugant, yielding normal heterozygotes.

Timing of induction of pronuclear fusion failure and self-fertilization: Both self-fertilization and fu- sion failure were induced when pairs were treated with vinblastine at 5 hr into conjugation. If these two alternate conjugational pathways result from the dis- ruption of different events ( k , pronuclear exchange and pronuclear fusion, respectively) one might expect that they would be induced by vinblastine at different periods during conjugation. Since these two events occur in quick succession (RAY 1955, 1956) the in-

Pronuclear Fusion Failure 633

"T I A

FIGURE 4.-Time course of induction of pronuclear fusion failure and self-fertilization by vinblastine. At various times after the start of mating samples of mating culture were treated with vinblastine (3 kg/ml, final concentration). After a half hour treat- ment the cells were diluted into water and centrifuged. The pellet was resuspended in water to give approximately a 1000-fold dilution of the drug. After exconjugants separated, they were isolated into drops of medium (ORIAS and HAMILTON 1979). The conjugation pathway of each exconjugant was identified by its pattern of drug resistance; presumptive self-fertilizations and fusion failures were testcrossed to confirm their micronuclear genotypes (see MATERIALS AND METHODS). Key to symbols: circles, pronuclear fusion failures; squares, self-fertilizations.

duction periods should be close. T o test this hypoth- esis mating cultures were treated with 30-min vin- blastine pulses throughout conjugation; the conjugation pathway followed by each exconjugant was identified from its phenotype (Table 3). All putative fusion failure caryonides were testcrossed so their micronuclear genotypes could be identified. Only a limited number of progeny (6-12) were isolated from these testcrosses but this was usually more than adequate for our purposes. All putative self-fertilizers were also testcrossed; those found to be heterokaryons were reclassified as fusion failures in which only one sister caryonide survived. Half of such clones should be heterokaryons and thus iden- tifiable by testcrosses. Thus testcrossing increases the level of accuracy of conjugation pathway assignment. Our experiments showed that the frequency of self- fertilization increased about 15 min earlier than that of fusion failure, with maxima at 5.25 and 5.5 hr, respectively. Figure 4 shows the results of a repre- sentative experiment.

DISCUSSION

Autoradiographic experiments suggested that it was possible to induce pronuclear fusion failure in T. ~ h e r m ~ p ~ 2 ~ u by giving mating cells a pulse treatment with vinblastine (HAMILTON and SUHR-JESSEN 1980; this paper). This study presents additional autora- diographic and genetic evidence for the occurrence of this conjugational abnormality. This process occurs when the incoming migratory pronucleus fails to fuse with the resident stationary pronucleus. Each nucleus independently undergoes division and differentia-

tion to produce a new macro- and micronucleus (Figure 2). Thus each exconjugant contains new nuclei of differing origins, and since each nucleus is derived from a single haploid meiotic product it is also a whole genome homozygote. The progeny generated by this process are usually viable with a normal diploid genome.

Our conclusions about fusion failure are based on the following genetic and autoradiographic lines of evidence. (1) Among the progeny of presumptive fusion failure pairs, sister caryonides differ in their macronuclear determined phenotypes. Furthermore, the macronuclei are quadruplyhomozygous for un- linked genes derived exclusively from different par- ents. (Sister caryonides are normally alike in macro- nuclear phenotype and micronuclear genotype, as the new nuclei of an exconjugant are usually derived from a single fertilization nucleus.) (2) Half of our fusion failure progeny are heterokaryons, their micro- and macronucleus being uniparental in origin, but each derived from a different parent. (3) Auto- radiography of pairs fixed after macronuclear de- velopment showed some conjugants with new nuclei of uniparental but mixed origins (Figure 1B).

Homozygosity after pronuclear exchange could be generated by genetic mechanisms other than fusion failure, e.g., crossing-over or chromosome nondis- junction during the postzygotic mitotic divisions. However, progeny which were heterozygous at some of the loci but homozygous at the others rarely, if ever, occurred. Such progeny should be generated by these mechanisms much more frequently than quadruple homozygotes, considering that Mpr, Chx, c d d and cduC are each on a different chromosome (BRUNS 1982). This argument plus the autoradi- ographic evidence make it extremely likely that pro- nuclear fusion failure is the correct interpretation. Spontaneous fusion failure has been observed cyto- logically in other ciliates [see RAIKOV (1972) for review] and mutations which cause an analogous fusion failure (Kur) have been found in yeast (CONDE and FINK 1976). Furthermore, microtubule disrupt- ing drugs have been shown to prevent pronuclear fusion in sea urchins (SCHATTEN and SCHATTEN 1981 ; SCHATTEN et ul. 1982; SCHATTEN 1984) and yeast (DELGADO and CONDE 1984).

Our results also provide the first genetic evidence that only one of the two new micronuclei which develop in each conjugant remains functional (Table 4), confirming previous cytological observations (NANNEY 1953; R A Y 1956). Normal vegetative T. thermophilu cells appear to be capable of reliably maintaining only one micronucleus. The destruction of one of the two micronuclei is random with regard to genotype and parental source; all three possible combinations of functional micronuclei (with regard to parental origin) are found among the sets of

634 E. P. Hamilton, P. B. Suhr-Jessen and E. Orias

exconjugants, and the ratio is close to the expected 1 : 2 : 1 (Figure 3).

How can the conjugants withstand the failure of such a key event as the fusion of gametic pronuclei and go on to produce viable progeny with normal nuclear complement? Four new nuclei will be gen- erated after fusion failure if the division which nor- mally succeeds pronuclear fusion is also inhibited by vinblastine and the two unfused nuclei take the place of the first postzygotic division nuclei. No further deviations from normal conjugation need be invoked. In the second postzygotic divisions the spindles are oriented along the longitudinal axis of each cell; this deposits the daughter nuclei at opposite ends of the cell. Local differences in cytoplasmic environment at each end then result in the differentiation of anterior nuclei into macronuclei, while posteriorly located ones become micronuclei (NANNEY 1953; R A Y 1956). If the unfused gametic pronuclei undergo this divi- sion each would give rise to a micro- and a macro- nucleus as predicted.

The normal micronuclear ploidy of the fusion failure progeny can also be explained without invok- ing an altered schedule of DNA replication. In most eukaryotes the round of DNA synthesis which pre- cedes the division of the fertilization nucleus occurs after the fusion of gametic pronuclei, but in Tetra- hymena this round has been advanced to the time before the reciprocal exchange of the gametic pron- uclei (DOERDER and DEBAULT 1975). Thus, the two pronuclei are already replicated and the division which occurs immediately after their fusion produces two diploid products. Blocking fusion and the sub- sequent division leaves exactly the same nuclear ploidy, as illustrated in Figure 5.

Vinblastine induces not only pronuclear fusion failure but also other conjugational abnormalities. Many of the treated pairs undergo self-fertilization (cytogamy); this results when the reciprocal exchange of gametic pronuclei is blocked (ORIAS and HAMILTON 1979; HAMILTON and SUHR-JESSEN 1980). In these pairs the sister gametic pronuclei may fuse (as usually happens with migratory and stationary pronuclei) or their fusion may also be blocked; our results cannot distinguish between these possibilities. The vinblas- tine treatment also induces other rarer genetic and cytological abnormalities, e.g., abnormal numbers of nuclei or macronuclear retention. Longer exposures to the drug (or higher concentrations) blocks the development of new macronuclei, as detected genet- ically (retention of the phenotype determined by the old macronucleus) or cytologically (our unpublished results).

It is as yet unknown how vinblastine, a known microtubule disrupting drug (WILSON, ANDERSON and CHIN 1976), causes self-fertilization and pronuclear fusion failure. Microtubules have been observed in

A B

x=== F

In 2c

FIGURE 5,”Simplest sequence of events that would account for the generation of diploid micronuclei after pronuclear fusion failure. Column A, Normal conjugation (pronuclear exchange prior to fusion) or self-fertilization (no exchange prior to fusion). Column B, Pronuclear fusion failure with suppressed first post- zygotic mitosis I. The same consequence is expected regardless of whether pronuclear exchange occurs or not. The second alternative represents a variation of self-fertilization. Each row shows the entire nuclear complement of a particular homologue, ploidy (n) and DNA amount (C). Circles and lines represent centromeres and DNA duplex, respectively. Open and solid centromeres are derived from different parent cells. S, DNA synthesis period; F, pronuclear fusion; CMD, centromere duplication; AP, anaphase; KK, karyokinesis; PZM, postzygotic mitosis. Dashed arrows suggest uncertainty concerning the timing of centromere duplication and generation of two independent chromosomes.

the areas that the pronuclei move through during exchange and fusion and have been implicated in other nuclear movements (AIST and WILLIAMS 1972; BEISSON and ROSSIGNOL 1975; WILLIAMS and WIL- LIAMS 1976; MESSIER 1978; WOLFE 1978; OAKLEY and MORRIS 1980; LANNERS 1980; ORIAS, HAMILTON and ORIAS 1983; DELCADO and CONDE 1984). The fact that many other microtubule disrupting drugs and a microtubule stabilizing drug (Taxol; our unpublished results) also induce these alternate conjugational pathways suggests that these drugs are acting through their specific effects on microtubules. The impor- tance of correct timing of treatment and the short sensitivity period suggest that vinblastine disrupts a microtubule system(s) specially assembled and/or maintained during conjugation for the fertilization events. The effect of vinblastine binding to its target appears to be reversible since the dilution which terminates treatment allows subsequent events to proceed normally. Continuous exposure of conju- gating cells to the drug blocks further conjugational and developmental steps (our unpublished results; KACZANOWSKI, GERTIC and KUBIAK 1985).

Hyperosmotic shock is another treatment which blocks the reciprocal transfer of gametic pronuclei (ORIAS, HAMILTON and FLACKS 1979). However, hy- perosmosis and vinblastine appear (at least in part) to differ in their mode of action, as hyperosmotic shock produces a much higher ratio of self-fertiliza-

Pronuclear Fusion Failure 635

tions to fusion failures (ORIAS and HAMILTON 1979; HAMILTON and SUHR-JESSEN 1980).

There are many immediate uses for our technique of inducing pronuclear fusion failure. A direct one is in the construction of heterokaryon strains which have genetically different macro- and micronuclei. These strains can be extremely useful in both genetic and biochemical studies and are now readily produ- cible by this one step method (MAYO and ORIAS 1980, 1985). The use of parents which are sensitive heter- okaryons for drug resistance markers (like those which allowed our genetic detection of fusion failure) facilitates heterokaryon construction by our method.

Another prospective use for our discovery is in the isolation of mutants defective in any aspect of the conjugation program controlled by the parental ma- cronucleus. The technical difficulties posed by this class of mutations are that (1) the phenotypic effects can only be detected in the F2 (after mutagenesis) and (2) molecules are efficiently exchanged between mates during conjugation (MCDONALD 1966; E. ORIAS, M. FLACKS and B. SATIR, personal communi- cation), so that both F1 mates must have macronuclei which are homozygous for the same mutation. These requirements are uniquely met by certain caryonides of a fusion failure pair, when appropriate drug- resistance markers are used in “trans” (as in the present study). A mixture of caryonides b and c (Figure 2) can be selected, using the recessive 2-dGal- resistance to select for their homozygous macronuclei. Remating can occur within this mixture because caryonidal mating types are independently deter- mined, and under the appropriate conditions gen- erally different (NANNEY 1956). After remating, prog- eny can be selected by using the drug resistance marker (e.g. , cycloheximide resistance) present in the micronucleus of caryonide c (Figure 2). If the original 2-dGal parental heterokaryon was mutagenized (prior to inducing fusion failure) then the conjugation of the two F1 clones can proceed under the direction of the two parental macronuclei which are homozy- gous for the same “conjugational program” mutation. Since both fusion failure and F2 progeny can be selected in turn, this approach can be adapted to efficient mechanical cloning, subculturing and cross- ing methods based on 96-well (microtiter) plate tech- nology which have already been used extensively in Tetrahymena genetics (ORIAS and BRUNS 1975; BRUNS and SANFORD 1978).

This work was supported in part by National Institutes of Health grant GM 19190 to E.O. and grants from the Danish National Science Research Council and the Carlsberg Foundation to P.S.-J.

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Communicating editor: S. L. ALLEN