Current Genetics
Current Genetics (1984) 8 : 3 4 5 - 3 5 2
© Springer-Verlag 1984
Instability of Saccharomyces cerevisiae heterokaryons Tahla Benltez I , Lucas del Castillo I , Andr6s Aguilera 1 , and Jaime Conde 2 1 Departamento de Gen6tica,Facultad de Biolog]a,Universidad de Sevilla,SeviUa 2 Cruzcampo, S.A., Sevilla,Spain
Summary. We have constructed heterokaryons of Saccharomyces cerevisiae by crossing k a r l - mutants incapable of nuclear fusion. Approximately 1% of the total zygotes from karl - crosses can form heterokaryotic clones. These are very small as compared to diploid colonies, and are composed mainly of a mixture of both types of heteroplasmons (cells which contain the cytoplasmic components of both parents, but the nuclear genotype of only one of them). This fact indicates that heterokaryons are unstable. This instability is already observed by pedigree analysis in the first zygotic divisions. We suggest that missegregation is the main factor in heterokaryon instability and results from an unequal nuclear transmission, which occurs when one of the mother nuclei divides and, although viable, does not migrate to the daughter bud. However, the proportion of inviable zygotes and buds found in the pedigree analysis, as well as the recovery of only one type of heteroplasmon, indicates the complete loss of one of the parental nuclei. Consequently nuclear inactivation is suggested as the second reason for heterokaryon instability. Key words: Yeast heterokaryons - Heterokaryon instability - Missegregation - Nuclear inactivation
Introduction
Mutations have been described which prevent nuclear fusion during the sexual cycle of Saccharomyces eerevisiae (Conde and Fink 1976; Polaina and Conde 1982; Dutcher and Hartwell 1982). Cytological studies reveal that in crosses in which one of the parental strain Offprint requests to: T. Ben~'tez
carries a k a r l - mutation, normal mating occurs but the subsequent karyogamy fails, resulting in a dikaryotic zygote. This zygote buds normally and the two nuclei divide giving rise to either cells containing only one of the parental nuclei but a mixed cytoplasm (heteroplasmons) or cells containing both of the parental nuclei and mixed cytoplasm (heterokaryons). These cells continue their propagation under appropriate conditions. Heterokaryons can ocassionally form synkaryons by fusion of their nuclei. A heterokaryon is not a natural phase in the life cycle of S. cerevisiae, but the result of a failure in the process of nuclear fusion. The presence in a cell of at least two nuclei for only one bud, brings about new situations when nuclear division takes place in the dikaryotic zygote; either both of the daughter nuclei migrate together to the zygotic bud or one of them remains in the zygote. This latter mechanism, which we have called missegregation, is a phenomenon observable by cytological methods in zygotes and vegetative cells, and supported by the existence of multinucleated cells in heterokaryotic colonies (Conde and Fink 1976). Its consequence is an unequal transmission of the nuclei to the daughter cells, producing a very small fraction of heterokaryotic cells and a variable fraction of each heteroplasmon (Dutcher 1981 ; Dutcher and Hartwell 1983a). Nuclear inactivation cannot be so clearly demonstrated as missegregation, although in heterokaryotic cells it seems to be a common phenomenon. Dutcher (1981) has reported on the high frequency of inviable cells found in haploid progeny emanating from heterokaryotic zygotes ofS. cerevisiae. In heterokaryons of Schizophyllum commune, Leonard et al. (1979, 1980) found that dedikaryotization into their monokaryotic components was associated with a genetic alteration of one of the component nuclei. The result was the recovery of only one nuclear type.
346
T. Benltez et al.: Instability of yeast heterokaryons
Table 1. S. cerevisiae strains used Strain
Source
Genotype
X51-2B X51-2C X51-1A X51-1D 4A A10701C A941B SL107-8B 4836-8C MC40 5464-7C JC25 ABQ21 F19 5307-13D ES2 F311/F13A10
J. Conde J. Conde J. Conde J. Conde J. Conde G. Fink G. Fink S. Liebman G. Fink G. Fink G. Fink G. Fink J. Polaina J. Conde G. Fink This study G. Fink
MA Ta leul karl-1 MATa his4-£xl5 ade2-1 karl-1 MATa karl-1 his4-A15 ade2-1 MA Ta karl-1 his4-/x15 leul MA Ta leu eth R MA Ta thr4 MA Ta trp 5 MATa leu2-1 metS-1 MA Ta leul thrl MA Ta/a lysl /lysl MATa his2 his4-38 adel trpl arg4 asp5 cdc14 MA Ta are2-1 his4-A15 karl-1 [KIL-K ] MA Ta his4-A15 ade2-1 canR nys R karl-2 MA Ta lys 2 MATa trpl ura4 met2 leu2 lys2 MATa leu2-1 eth R karl-1 MATa leu2-1 eth R
In this w o r k w e have s t u d i e d the fate o f t h e c o m p o n e n t nuclei during t h e f o r m a t i o n a n d f u r t h e r divisions o f y e a s t h e t e r o k a r y o n s . Our c o n c l u s i o n is t h a t b o t h miss e g r e g a t i o n a n d n u c l e a r i n a c t i v a t i o n a c c o u n t for h e t e r o k a r y o n instability.
Materials a n d m e t h o d s
Yeast strains and genetic methods. S. cerevisiae strains used in this work are listed in Table 1. ES2 is an isogenic strain obtained after crossing the strains ABQ21 and F 311/F 13A10, and sporulating the cross. A meiotic product of such a cross was backcrossed with the parental ABQ21. This new cross, once sporulated, gave rise to ES2 and several other 50% isogenic strains. Other strains with various genotypes and 97% isogenicity were obtained by repeating the backcross five times. The genotypic designations karl-l, K A R l , [RHO÷], [RHO-], [RHO-o], [KIL-Kland [KIL-o] have been already described (Conde and Fink 1976); eth R, canR and nys R are designations for phenotypes caused by recessive mutations which confer resistance to ethionine, canavanine and nystatine respectively. The genetic methods are those described in the Cold Spring Harbor Yeast Course Manual (Sherman et al. 1979, unless otherwise stated. Culture media. Complete medium (YPD),'minimal medium (SD) and sporulation medium (SPO), have been described (Sherman et al. 1979. To supplement auxotrophic requirements we added the appropriate aminoacids or bases to minimal medium (Sherman et al. 1979. Canavanine was used at a final concentration of 60 rag/1 with haploids and 120 mg/1 with diploids, ethionine at 1 mg/l and nystatin at 2 mg/1. Terminology. The nuclei of the mating strains are called P1 and P2 respectively; in the pedigree analysis, the first three micromanipulated buds from the zygote are called H 1, H 2 and H3, respectively. The first bud micromanipulated from H 1 is called H1.1 . We differentiate between heterokaryotic zygotes (P1 * P2), in which nuclear fusion has failed, and synkaryotic ones (P1/P2) in which both nuclei have fused. Heterokaryons can be identified
because when placed on rich medium, they plasmons, which have P1 or P2 nuclei but mixed. Synkaryotic zygotes are unable to plasmons, they can sporulate and are unable to
segregate heterothe cytoplasm is segregate heteroconjugate.
Pedigree analysis: 1. Zygotes and their first three buds (H I, H 2 and H3}. This pedigree analysis was carried out by mieromanipulating zygotes with a central bud. The mating mixture was incubated for 3 - 4 h at 30 °C on solid YPD. The zygote and the first three buds H1, H 2 and H 3 were micromanipulated; pedigree analysis was carried out on two different media: on SD minimal to detect heterokaryon stability and on YPD to determine the genotype of each clone. Once the cells grew on YPD, we replicated the clones onto the appropriate selective media. For example, assuming that the parental strains harbor complementarity auxotrophic and recessive resistance markers, and that heterokaryons complement the way synkaryons do (Dutcher and Hartwe11 1983a), we expected the following responses to the growth tests:
Phenotype of the clone
Media SD
SD +auxl + anx2 + drugl
SD + aux3 + anx4 + drug2
Heteroplasmons (P1) (auxl aux2 drug1 r)
-
+
-
Heteroplasmons (P2) (aux3 aux4 drug2 r)
-
-
+
Heterokaryons (P1 * P2) a
+
+
+
Synkaryons (P1/P2)
+
-
-
A heterokaryotic cell would be unable to grow in any media but SD. However, a heterokaryotic colony can grow in SD as well as in the two selective media because these colonies are a mixture of P 1, P2 and P1 * P2 cells
T. Benltez et al. : Instability of yeast heterokaryons
347 2. Zygotes, their first bud (HI} and the first bud of H I (HI.I). Zygotes with central buds were micromanipulated. These buds were separated from the zygotes by micromanipulation and allowed to produce new buds. These were separated again when bud formation had finished. Pedigree analysis took place on YPD. Once the cells grew on rich medium we replicated the clones onto the appropriate media as before. Characterization of heterokaryon colonies. Effect of the genetic background. Heterokaryon colonies were analyzed and the resuits compared to those of the pedigree analysis. We studied the progeny obtained after crossing three different k a r l - mutants with then strains which have several complementary auxotrophies and drug resistances, and calculated for each cross: 1) The percentage of zygotes to see mating efficiency. 2) The percentage of heterokaryons and synkaryons, to determine the leakiness of the k a r l - mutation and the viability and stability of the cross. And 3) The number of heteroplasmons of each type, to detect whether missegregation and nuclear inactivation are random processes or there is a bias against certain nuclei. Colony analysis was carried out by spreading a mating mixture on solid minimal medium (SD). The mating strains were previously grown for two days on solid YPD at 30 °C and then mixed and incubated for 3 h on solid YPD at 30 °C. The percentage of zygotes was calculated by counting 300 cells under the microscope. To determine the genotype of the selected colonies appearing on solid SD, we prepared suspensions of several independent colonies and these were spread on rich medium, minimal medium and medium supplemented with the requirements of one or the other nucleus. Nuclear staining. Fixed cells were stained with Giemsa according to the modified procedure of Robinow (HartweU et al. 1970).
Results Cytological observations o f heterokaryons
Fig. I A - F . karl - x karl - heterokaryons stained with Giemsa. A Vegetative cells, most of them binucleate. The nuclei are close to each other and situated between the big vacuole and the cell wall. This position probably contributes to a decrease in missegregation frequency. B Two nuclei are dividing synchronously in the bridge between mother and daughter cells. C Two nuclei are dividing synchronously but only one is in the division bridge (missegregation). D Polynucleated cell arising from missegregation. E Cell with an odd number of nuclei, suggesting that unequal nuclear transmission has already taken place. F Lack of synchrony between the two nuclei could give rise to unequal nuclear transmission. In this picture only one of the two nuclei in a dikaryon is dividing. A, B and D, x 1,080;C, x 1,800;E and F, x 2,160
H e t e r o k a r y o t i c colonies were obtained by making crosses b e t w e e n several Kar+ and K a r - strains and selecting t h e m on minimal m e d i u m as described in Methods. Only 1 - 3 % o f the colonies were heterokaryons, the rest o f t h e m being synkaryons. The h e t e r o k a r y o n s were clearly distinguishable because o f their c o l o n y size, highly reduced as c o m p a r e d to synkaryotic colonies. Their h e t e r o k a r y o t i c nature was checked by growth tests and nuclear staining. In spite o f the low f r e q u e n c y o f h e t e r o k a r y o t i c colonies and their reduced size, which could indicate high instability, most o f the stained vegetative cells o f these colonies w h e n freshly isolated have t w o nuclei, close to each o t h e r (Fig. 1A). Three situations were observed on nuclear division: either b o t h nuclei were transferred to the daughter cell (Fig. 1B) or one o f the nuclei was transferred, whereas the o t h e r divided at the opposite end o f the b u d ' s neck, producing uni- and trinucleated cells respectively (missegregation) (Fig. 1C). This m e c h a n i s m gives rise eventually to plurinucleated cells having f r o m t w o to t e n nuclei (Fig. 1D, E). A third observation was asynchronous
348
T. Benltez et al.: Instability of yeast heterokaryons
Table 2. Pedigree analysis during the first three divisions of 60 zygotes from the cross X51-1A (PI) x X51-1D (P2) Genotype of the clones
Buds
P1 heteroplasmons P2 heteroplasmons P1 * P2 heterokaryons P1/P2 synkaryons Non viable
Table 4. Pedigree analysis during the first division of 152 zygotes and the first division of H 1 buds, from the cross X51-2B (P2) x X51-2C (P1)
Zygote
H1
H2
H3
16 24 8 2 10
14 27 4 2 13
10 18 6 2 24
16 16 12 2 14
Genotype of the clones
Zygotes
H 1 bud
HI_ 1 bud
P1 heteroplasmons P2 heteroplasmons P1 * P2 heterokaryons P1/P2 synkaryons Non viable
50 35 27 5 35
70 37 13 5 27
75 45 8 5 19
152
152
152
Total
Table 3. Pedigree analysis of 21 fully viable zygotes and their buds from the cross X51-1A (PI) x X51-1D (P2) Type of zygotes
Number of zygotes
Type of buds H1
H2
H3
2 2 1 1 1
P2 P1 * P2 P1 * P2 PI*P2 P2
P2 P2 P2 P1 P1
P2 PI P1 * P2 PI *P2 PI
3 1 1 1
P2 P2 P1 * P2 P1
P2 P2 P2 P1 * P2
P1 * P2 P2 P2 P1
P2
2 1 1 1 1
P2 PI * P2 P1 P1 P1
P2 P2 P2 P1 * P2 PI
P2 P2 P2 P2 P2
Total
6
P1/P2
2
PI/P2
P1/P2
PI/P2
Total
21
PI *P2
Total P1
Total
divisions so that inside the same cell, one nucleus was dividing while the other was not (Fig. 1F).
Heterokaryon instability detected by pedigree analysis 1. Zygotes and their first three buds. In these experiments the strains X51-1A and X51-1D listed in the Table 1 were used.
a) Pedigree analysis on SD: In this experiment only H1 (central buds) were micromanipulated; 10 out of 110 zygotes analyzed were able to grow on SD. Of these, 5 were synkaryons and 5 heterokalyons. This frequency of synkaryons coincides with that expected from the leanness of the k a r l - mutation (Conde and Fink 1976). b) Pedigree analysis on YPD: H1, Hz and H3 buds were micromanipulated from 60 zygotes; H1 were always central buds; H2 and H 3 emerged from the same lateral side in 17 cases; from opposite lateral sides in 24 cases and one of them was central in 4 cases. The results obtained after analysing the colonies grown on YPD are shown in Table 2. There is a very high proportion of P1 and P2 heteroplasmons as compared to the number of heterokaryons. In the heteroplasmonic zygotes the number of P1 heteroplasmons is equal to that of P2 heteroplasmons, while in the first three buds H 1, H2 and H 3 this number is significantly higher for P2 heteroplasmons. This result indicates that unequal transmission of the nuclei to the buds is taking place. The viability of the micromanipulated buds decreases in such a way that H3 is less viable than He and this less than H1. The frequency of synkaryons observed in Table 2 does not differ from what was expected. There are two cases in which all the buds H1, H2 and H 3 are synkaryons. In these two situations the zygotes are also synkaryons. Table 3 shows the frequencies of different pedigree types for those cases in the previous experiment where both the zygote and the three zygotic buds, H1, H2 and H3, were fully viable. As in Table 2, the number ofheteroplasmonic P1 zygotes is equal to that of P2 zygotes, while in the buds the number of P2 heteroplasmons is higher than that of PI type. Unequal nuclear transmission is reinforced when one compares the ratio PI * P2 versus P1 + P2 of zygotic clones to that of the three zygotic buds (Table 3): 7 (Pl * P2) versus 12 (P1 orP2) for the zygotes, and 13
T. Benltez et al.: Instability of yeast heterokaryons
349
Table 5. Pedigree analysis of 100 fully viable zygotes, their first bud (HI) and the first bud of H 1 from the cross X51-2B (P2) x
X51-2C (P1) A Type of Zygotes
B Number of zygotes
P1 * P2
13 9 2 1
Total
25
P1
34 5 3 3
Total
45
P2
14 7 2 1 1
Total
25
P1/P2
5
Total
100
Type of buds
Type of buds H 1
H1
HI.1
P1 P2 P1 *P2 P2
P1 P2 PI*P2 P1
P1 P2 P1 * P2 P1 * P2
P2 P1 P1 * P2 P1 * P2 P1
P1/P2
P1 P2 P1 * P2 P1
P2 P1 P2 P1 * P2 P2
P1/P2
P1 * P2
Number of buds H 1
3 2 6
Total
11
P1
54 1
Total
55
P2
28 1
Total
29
P1/P2
5
Total
100
Type of buds H 1-1
P1 P2 P1 *P2
P1
P2
P2 P1
P1/P2
Data are independently organized according to the type of zygote A and to the type of H 1 bud B
(P1 * P2) versus 44 (P1 or P2) for the buds. These ratios are significantly different at the 95% level.
2. Zygotes, their first bud (111) and the first bud of H1 (1t1.1). We wanted to check if heterokaryon instability was due to a phenomenon occurring mainly in the zygotes. Such being the case, one can predict that, once the two nuclei have been transferred to the daughter cell, the probability of their being maintained through consecutive divisions should increase. To test this we have done a pedigree analysis in 189 zygotes. In these experiments the strains X51-2B and X51-2C listed in Table 1 were use:l. Zygotes, HI and H H buds were micromanipulated on YPD as described in Methods. The results are shown in Table 4. Only those zygotes from which it was feasible to separate the H I bud and from this the HI.1 bud are shown (152 zygotes). The proportion of P1 and P2 heteroplasmons is even higher than that of the previous experiment (Table 2). Also,
the relative numbers of P1 and P2 heteroplasmons indicate that the nuclei are not transferred with the same frequency. Frequencies of nuclear fusion (synkaryons) are comparable to previous results (Table 2). Table 5 shows the frequency with which heteroplasmons of each type, heterokaryons or synkaryons, are segregated when the zygotes and their buds H1 and Ha.1 are fully viable (100 zygotes). In this Table, as in Table 4, the unequal nuclear transmission is very accentuated (45 PI zygotes versus 25 P2 zygotes; 55 P1, H1 buds versus 29 P2, H1 buds; 58 P1, Hi-1 buds versus 31 P2, H1-1 buds). Out of 100 zygotes there are 34 and 14 colonies where the zygotes as well as the H 1 and the H1.1 buds are respectively P1 or P2, while the other nucleus has disappeared. There is also a fairly high frequency of inviable zygotes (Table 4). Unequal nuclear transmission is indicated by the 2 5 - 7 0 ratio of P1 * P2 versusPl + P2 in zygote colonies compared to the 11-84 ratio in H 1 colonies.
350
T. Benltez et al. : Instability of yeast heterokaryons
Table 6. Percentage of heterokaryon colonies obtained from the crosses described Mating strains a
Mating strains a
4A A10701C A941B SL107-8B 4836-8C MC40 5464-7C
JC25
ABQ21
14 15 14 20 13 8 10
9 12 12 12 20 4 16
percentage o f heterokaryons was even lower than in normal crosses. The results of heteroplasmon segregations did not differ between both types of crosses (data n o t shown). The effect o f isogenicity upon heterokaryon stability was also studied b y analyzing clones derived from crosses between isogenic strains. In spite o f an increasing degree o f isogenicity (up to 97%) stable heterokaryons were not obtained in any o f the crosses studied (data n o t shown).
Discussion
The results are the average of three experiments
1. Missegregation and nuclear inactivation as causes of heterokaryon instability Missegregation and nuclear inactivation in heterokaryon colonies: effect of the genetic background Table 6 shows the percentage o f heterokaryon colonies obtained in the different crosses. These percentages are n o t significantly different except in the crosses involving MC40, an a/a diploid strain. Table 7 shows the distribution o f synkaryons, heterokaryons and heteroplasmons, in the heterokaryon colonies analyzed from each cross. Auxotrophies, resistances or any other differences in the genetic backgrounds do n o t seem to affect the distribution o f the different genotypes, except in the cross with the strain MC40, the only diploid strain tested, which shows a very strong polarity in favor o f the diploid parental nucleus. The effect o f heterozygosis for mating type upon heterokaryon stability was studied. When crosses between strains o f the same mating type were tested, the
Our results indicate that heterokaryons are extremely unstable entities. The nuclei are not equally transmited from the mother to the daughter cells, due to two phenomena, missegregation and nuclear inactivation. The results that support missegregation are the following: a) Cytological observations (Fig. 1C). b) The frequency o f cases in Table 5 where zygotes are heterokaryons and H 1 buds heteroplasmons (22 lineages) compared with those where zygotes are heteroplasmons and HI buds heterokaryons (10 lineages). These frequencies should be similar if instability were only due to nuclear inactivation, c) The proportion of P1 and P2 heteroplasmons in the zygotes compared with that in the H1, H2 or H 3 buds (Table 2). Were nuclear inactivation the only reason for the appearance of heteroplasmons, the ratio should be the same in the zygote as in the H1, H2 and H 3 buds, which is not the case.
Table 7. Number of heterokaryon, heteroplasmon, and diploid clones obtained from heterokaryon colonies Mating strains
Mating strains (P1) JC25
ABQ21
(P2)
Heterokaryon P1 * P2
Synkaryons P1/P2
Heteroplasmon P1
P2
MC40 4A SL107-8B 4836-8C A10701C A941B
795 980 208 604 282 178
57 228 19 163 10 20
290 401 363 848 676 208
957 670 160 1,200 880 324
Total
1,820 1,302 531 3,316 1,028 641
Heterokaryon P1 * P2
Synkaryons P1/P2
208 138 328 478 55 667
44 13 7 30 0 75
Heteroplasmon P1
P2
140 156 565 104 120 1,896
448 307 424 278 130 1,962
Total
928 503 881 652 177 4,041
The heterokaryon colonies are mixtures of heteroplasmons, heterokaryons and synkaryons. Since a single colony may have heteroplasmons of each type, as well as heterokaryons and synkaryons, the total number of colonies is not the addition of the previous four colums. Data given are the results of three experiments. 9 independent colonies were analyzed in each experiment
T. Benitez et al.: Instability of yeast heterokaryons The existence of nuclear inactivation is supported by the following results: a) The high frequency of pedigrees in which a mother cell is heteroplasmonic while the corresponding daughter cell is heterokaryotic (Table 3 and 5). This cannot be explained only by missegregation, b) The existence of pedigrees with only one type of nucleus while the other nucleus has disappeared completely. In Table 3, the zygotes as well as H x , H2 and H 3 buds are all P2, in 2 cases. In Table 5, this happens in 34 cases for the P1 nucleus and 14 cases for the P2 nucleus, c) The high percentage of inviable zygotes, 25% in Table 2 and 23% in Table 4. d) Those cases in which both mother and daughter cells are heteroplasmons, but one of them is P1 while the other is P2 (Tables 3 and 5). e) Those cases in which there is a strong polarity in favor of one of the nuclei when this nucleus is 2n (Table 7).
351 Scheme 1
Zygotes
tll buds
HI_I buds
P1 or P2
P1 or P2
82 85%//t
/ Prior to first mitosis:
PI*P2
After first mitosis:
PI*P2 25
/
15% , PI*P2
95 l
7 54% / I
13 26%
46% ,
PI*P2 6
185% PI*P2 11
2. Differences in stability between heterokaryotic zygotes and heterokaryotic vegetative cells
3. Influence of the genetic background
Heterokaryon instability is present both in zygotes and in vegetative cells, although it is stronger in the former. The high frequency of heteroplasmons, which segregate even in the first zygotic bud (Table 2), together with the low frequency of heterokaryotic colonies (about 1% of the zygotes) and the size of these colonies, very small when compared to synkaryon colonies, led to the expectation of a frequency of heterokaryons among vegetative cells lower than it actually was. This means taht a strong selection against heterokaryons could already take place in the zygote, prior to or during the formation of the first zygotic bud. Once the first bud had both nuclei, the probability that they would be maintained in the cells seemed to increase. This could account for the fairly high fraction of heterokaryotic cells inside heterokaryon colonies (Fig. 1A and Table 7). To determine such differences in stability between heterokaryotic zygotes and vegetative cells we have compared the frequency with which a heterokaryotic cell is able to produce another heterokaryotic cell. In Table 5 it can be seen that only 3 out of 25 heterokaryotic zygotes produce heterokaryotic buds, whereas 11 heterokaryotic HI cells produce 6 heterokaryotic ones. Besides, from 100 viable zygotes from which the H 1 and H1-1 buds were also fully viable, we obtained the percentages of heterokaryons and heteroplasmons (shown in scheme 1), the frequency of transmission for both nuclei is 15% from the zygote to the first bud while the percentage is increased to 46% from this cell to its subsequent bud. Also, 26% of the zygotes continue to be heterokaryon after producing their first bud (H1). This frequency is 85% for the H 1 cell after producing its first subsequent bud. We therefore conclude that heterokaryosis is more stable in vegetative cells than it is in zygotes.
Another point to be discussed is the fortuitousness of the unequal transmission of the nuclei, as far as the genetic background (auxotrophies, drug resistances, sexual locus, isogenicity) is concerned. In Neurospora, homosexuality is an essential condition for obtaining stable heterokaryons (Gamjobst and Wilson 1956). We investigated if this was also the case for Saccharomyces, but our results did not support the hypothesis. Moreover, the number of heterokaryons obtained was lower than that obtained in heterosexual crosses. Nuclear inactivation could be due to a lack of identity in alleles of incompatible genes, as in Physarum (Carlile 1976). To test this hypothesis, strains of near isogenicity were constructed by means of five consecutive backcrosses (96,87% isogenicity). Nevertheless, heterokaryons were as unstable as those of non4sogenic strains. It is noteworthy that nuclear transmission is strongly polarized when a diploid nucleus is involved, and that it is the diploid nucleus the one which is preferentially transmited. Dutcher and Hartwell (1981, 1983a, b) have demonstrated that some gene products function both in nuclear division and in nuclear fusion, so that the lesion producing the defect in karyogamy is probably identical to the lesion producing the defect in cell division. If abortive mitosis occurs and some chromosomes are lost (Dutcher 1981), one should expect nuclear inactivation more frequently in haploid nuclei, because when a diploid nucleus is involved, the loss of a single chromosome would not lead to lethality, but only to an~uploid formation. The high viability of MC40 nuclei found in our results, as well as the very strong polarity in only this cross, support this point. Missegregation could occur because some type of clock controls-the bud emergence event, maintaining
352 only a single bud every cell cycle, and DNA synthesis and nuclear division can occur in the absence of bud emergence (Byers and Goetsch 1975). The nucleus in close proximity to the site of budding will migrate, producing a heteroplasmon, while the other one will divide, generally giving rise to a cell with three nuclei. This will explain the polynucleated cells found in vegetative cultures and zygotes (Figs. 1D and 1E). We have detected synkaryotic buds. only in the cases where the zygote was already a synkaryon. This means that nuclear fusion is a very precise event in the sexual cycle and secondary nuclear fusion hardly ever takes place. We conclude from our work that heterokaryon instability is a consequence of the structural and cell cycle limitations of the yeast cell, and then could hardly be improved.
T. Benitez et al.: Instability of yeast heterokaryons References Byers B, Goetsch L (1975) J Bacteriol 124:511-523 Carlile MJ (1976) J Gen Microbio193:371-376 Conde J, Fink GR (1976) Proc Natl Acad Sci USA 75:36513655 Dutcher SK (1981) Mol Cell Biol 1 : 245-253 Dutcher SK, Hartwell LH (1982) Genetics 100:175-184 Dutcher SK, HartweU LH (1983a) Mol Cell Biol 3 : 1255-1265 Dutcher SK, Hartwell LH (1983b) Cell 33:203-210 Garnjobst L, Wilson JF (1956) Proc Natl Acad Sci USA 42:613618 Hartwell LH, Culotti J, Reid B (1970) Proc Natl Acad Sci USA 66:352-359 Leonard TJ, Dick S, Gaber RF (1979) Genetics 88:13-26 Leonard TJ, Gaber RF, Dick S (1980) Genetics 89:685-693 Polaina J, Conde J (1982) Mol Gen Genet 186:253-258 Sherman F, Fink GR, Lawrence CW (1979) Methods in yeast genetics. Laboratory Manual. Cold Spring Harbor Laboratory, New York
Acknowledgements. We thank M. I. Carretero, A. Ferm[ndez and D. Su~rez for their skillful assistance, and E. Cerd~i, J. Polaina, I. L. Calder6n and K. Grohmann for helpful discussion. Financial support came for the "Comisi6n Asesora para la Investigaci6n Cientifica y Tecnol6gica" of the Spanish Government.
Communicated by B. S. Cox Received January 22, 1984
Current Genetics (1984) 8 : 3 4 5 - 3 5 2
© Springer-Verlag 1984
Instability of Saccharomyces cerevisiae heterokaryons Tahla Benltez I , Lucas del Castillo I , Andr6s Aguilera 1 , and Jaime Conde 2 1 Departamento de Gen6tica,Facultad de Biolog]a,Universidad de Sevilla,SeviUa 2 Cruzcampo, S.A., Sevilla,Spain
Summary. We have constructed heterokaryons of Saccharomyces cerevisiae by crossing k a r l - mutants incapable of nuclear fusion. Approximately 1% of the total zygotes from karl - crosses can form heterokaryotic clones. These are very small as compared to diploid colonies, and are composed mainly of a mixture of both types of heteroplasmons (cells which contain the cytoplasmic components of both parents, but the nuclear genotype of only one of them). This fact indicates that heterokaryons are unstable. This instability is already observed by pedigree analysis in the first zygotic divisions. We suggest that missegregation is the main factor in heterokaryon instability and results from an unequal nuclear transmission, which occurs when one of the mother nuclei divides and, although viable, does not migrate to the daughter bud. However, the proportion of inviable zygotes and buds found in the pedigree analysis, as well as the recovery of only one type of heteroplasmon, indicates the complete loss of one of the parental nuclei. Consequently nuclear inactivation is suggested as the second reason for heterokaryon instability. Key words: Yeast heterokaryons - Heterokaryon instability - Missegregation - Nuclear inactivation
Introduction
Mutations have been described which prevent nuclear fusion during the sexual cycle of Saccharomyces eerevisiae (Conde and Fink 1976; Polaina and Conde 1982; Dutcher and Hartwell 1982). Cytological studies reveal that in crosses in which one of the parental strain Offprint requests to: T. Ben~'tez
carries a k a r l - mutation, normal mating occurs but the subsequent karyogamy fails, resulting in a dikaryotic zygote. This zygote buds normally and the two nuclei divide giving rise to either cells containing only one of the parental nuclei but a mixed cytoplasm (heteroplasmons) or cells containing both of the parental nuclei and mixed cytoplasm (heterokaryons). These cells continue their propagation under appropriate conditions. Heterokaryons can ocassionally form synkaryons by fusion of their nuclei. A heterokaryon is not a natural phase in the life cycle of S. cerevisiae, but the result of a failure in the process of nuclear fusion. The presence in a cell of at least two nuclei for only one bud, brings about new situations when nuclear division takes place in the dikaryotic zygote; either both of the daughter nuclei migrate together to the zygotic bud or one of them remains in the zygote. This latter mechanism, which we have called missegregation, is a phenomenon observable by cytological methods in zygotes and vegetative cells, and supported by the existence of multinucleated cells in heterokaryotic colonies (Conde and Fink 1976). Its consequence is an unequal transmission of the nuclei to the daughter cells, producing a very small fraction of heterokaryotic cells and a variable fraction of each heteroplasmon (Dutcher 1981 ; Dutcher and Hartwell 1983a). Nuclear inactivation cannot be so clearly demonstrated as missegregation, although in heterokaryotic cells it seems to be a common phenomenon. Dutcher (1981) has reported on the high frequency of inviable cells found in haploid progeny emanating from heterokaryotic zygotes ofS. cerevisiae. In heterokaryons of Schizophyllum commune, Leonard et al. (1979, 1980) found that dedikaryotization into their monokaryotic components was associated with a genetic alteration of one of the component nuclei. The result was the recovery of only one nuclear type.
346
T. Benltez et al.: Instability of yeast heterokaryons
Table 1. S. cerevisiae strains used Strain
Source
Genotype
X51-2B X51-2C X51-1A X51-1D 4A A10701C A941B SL107-8B 4836-8C MC40 5464-7C JC25 ABQ21 F19 5307-13D ES2 F311/F13A10
J. Conde J. Conde J. Conde J. Conde J. Conde G. Fink G. Fink S. Liebman G. Fink G. Fink G. Fink G. Fink J. Polaina J. Conde G. Fink This study G. Fink
MA Ta leul karl-1 MATa his4-£xl5 ade2-1 karl-1 MATa karl-1 his4-A15 ade2-1 MA Ta karl-1 his4-/x15 leul MA Ta leu eth R MA Ta thr4 MA Ta trp 5 MATa leu2-1 metS-1 MA Ta leul thrl MA Ta/a lysl /lysl MATa his2 his4-38 adel trpl arg4 asp5 cdc14 MA Ta are2-1 his4-A15 karl-1 [KIL-K ] MA Ta his4-A15 ade2-1 canR nys R karl-2 MA Ta lys 2 MATa trpl ura4 met2 leu2 lys2 MATa leu2-1 eth R karl-1 MATa leu2-1 eth R
In this w o r k w e have s t u d i e d the fate o f t h e c o m p o n e n t nuclei during t h e f o r m a t i o n a n d f u r t h e r divisions o f y e a s t h e t e r o k a r y o n s . Our c o n c l u s i o n is t h a t b o t h miss e g r e g a t i o n a n d n u c l e a r i n a c t i v a t i o n a c c o u n t for h e t e r o k a r y o n instability.
Materials a n d m e t h o d s
Yeast strains and genetic methods. S. cerevisiae strains used in this work are listed in Table 1. ES2 is an isogenic strain obtained after crossing the strains ABQ21 and F 311/F 13A10, and sporulating the cross. A meiotic product of such a cross was backcrossed with the parental ABQ21. This new cross, once sporulated, gave rise to ES2 and several other 50% isogenic strains. Other strains with various genotypes and 97% isogenicity were obtained by repeating the backcross five times. The genotypic designations karl-l, K A R l , [RHO÷], [RHO-], [RHO-o], [KIL-Kland [KIL-o] have been already described (Conde and Fink 1976); eth R, canR and nys R are designations for phenotypes caused by recessive mutations which confer resistance to ethionine, canavanine and nystatine respectively. The genetic methods are those described in the Cold Spring Harbor Yeast Course Manual (Sherman et al. 1979, unless otherwise stated. Culture media. Complete medium (YPD),'minimal medium (SD) and sporulation medium (SPO), have been described (Sherman et al. 1979. To supplement auxotrophic requirements we added the appropriate aminoacids or bases to minimal medium (Sherman et al. 1979. Canavanine was used at a final concentration of 60 rag/1 with haploids and 120 mg/1 with diploids, ethionine at 1 mg/l and nystatin at 2 mg/1. Terminology. The nuclei of the mating strains are called P1 and P2 respectively; in the pedigree analysis, the first three micromanipulated buds from the zygote are called H 1, H 2 and H3, respectively. The first bud micromanipulated from H 1 is called H1.1 . We differentiate between heterokaryotic zygotes (P1 * P2), in which nuclear fusion has failed, and synkaryotic ones (P1/P2) in which both nuclei have fused. Heterokaryons can be identified
because when placed on rich medium, they plasmons, which have P1 or P2 nuclei but mixed. Synkaryotic zygotes are unable to plasmons, they can sporulate and are unable to
segregate heterothe cytoplasm is segregate heteroconjugate.
Pedigree analysis: 1. Zygotes and their first three buds (H I, H 2 and H3}. This pedigree analysis was carried out by mieromanipulating zygotes with a central bud. The mating mixture was incubated for 3 - 4 h at 30 °C on solid YPD. The zygote and the first three buds H1, H 2 and H 3 were micromanipulated; pedigree analysis was carried out on two different media: on SD minimal to detect heterokaryon stability and on YPD to determine the genotype of each clone. Once the cells grew on YPD, we replicated the clones onto the appropriate selective media. For example, assuming that the parental strains harbor complementarity auxotrophic and recessive resistance markers, and that heterokaryons complement the way synkaryons do (Dutcher and Hartwe11 1983a), we expected the following responses to the growth tests:
Phenotype of the clone
Media SD
SD +auxl + anx2 + drugl
SD + aux3 + anx4 + drug2
Heteroplasmons (P1) (auxl aux2 drug1 r)
-
+
-
Heteroplasmons (P2) (aux3 aux4 drug2 r)
-
-
+
Heterokaryons (P1 * P2) a
+
+
+
Synkaryons (P1/P2)
+
-
-
A heterokaryotic cell would be unable to grow in any media but SD. However, a heterokaryotic colony can grow in SD as well as in the two selective media because these colonies are a mixture of P 1, P2 and P1 * P2 cells
T. Benltez et al. : Instability of yeast heterokaryons
347 2. Zygotes, their first bud (HI} and the first bud of H I (HI.I). Zygotes with central buds were micromanipulated. These buds were separated from the zygotes by micromanipulation and allowed to produce new buds. These were separated again when bud formation had finished. Pedigree analysis took place on YPD. Once the cells grew on rich medium we replicated the clones onto the appropriate media as before. Characterization of heterokaryon colonies. Effect of the genetic background. Heterokaryon colonies were analyzed and the resuits compared to those of the pedigree analysis. We studied the progeny obtained after crossing three different k a r l - mutants with then strains which have several complementary auxotrophies and drug resistances, and calculated for each cross: 1) The percentage of zygotes to see mating efficiency. 2) The percentage of heterokaryons and synkaryons, to determine the leakiness of the k a r l - mutation and the viability and stability of the cross. And 3) The number of heteroplasmons of each type, to detect whether missegregation and nuclear inactivation are random processes or there is a bias against certain nuclei. Colony analysis was carried out by spreading a mating mixture on solid minimal medium (SD). The mating strains were previously grown for two days on solid YPD at 30 °C and then mixed and incubated for 3 h on solid YPD at 30 °C. The percentage of zygotes was calculated by counting 300 cells under the microscope. To determine the genotype of the selected colonies appearing on solid SD, we prepared suspensions of several independent colonies and these were spread on rich medium, minimal medium and medium supplemented with the requirements of one or the other nucleus. Nuclear staining. Fixed cells were stained with Giemsa according to the modified procedure of Robinow (HartweU et al. 1970).
Results Cytological observations o f heterokaryons
Fig. I A - F . karl - x karl - heterokaryons stained with Giemsa. A Vegetative cells, most of them binucleate. The nuclei are close to each other and situated between the big vacuole and the cell wall. This position probably contributes to a decrease in missegregation frequency. B Two nuclei are dividing synchronously in the bridge between mother and daughter cells. C Two nuclei are dividing synchronously but only one is in the division bridge (missegregation). D Polynucleated cell arising from missegregation. E Cell with an odd number of nuclei, suggesting that unequal nuclear transmission has already taken place. F Lack of synchrony between the two nuclei could give rise to unequal nuclear transmission. In this picture only one of the two nuclei in a dikaryon is dividing. A, B and D, x 1,080;C, x 1,800;E and F, x 2,160
H e t e r o k a r y o t i c colonies were obtained by making crosses b e t w e e n several Kar+ and K a r - strains and selecting t h e m on minimal m e d i u m as described in Methods. Only 1 - 3 % o f the colonies were heterokaryons, the rest o f t h e m being synkaryons. The h e t e r o k a r y o n s were clearly distinguishable because o f their c o l o n y size, highly reduced as c o m p a r e d to synkaryotic colonies. Their h e t e r o k a r y o t i c nature was checked by growth tests and nuclear staining. In spite o f the low f r e q u e n c y o f h e t e r o k a r y o t i c colonies and their reduced size, which could indicate high instability, most o f the stained vegetative cells o f these colonies w h e n freshly isolated have t w o nuclei, close to each o t h e r (Fig. 1A). Three situations were observed on nuclear division: either b o t h nuclei were transferred to the daughter cell (Fig. 1B) or one o f the nuclei was transferred, whereas the o t h e r divided at the opposite end o f the b u d ' s neck, producing uni- and trinucleated cells respectively (missegregation) (Fig. 1C). This m e c h a n i s m gives rise eventually to plurinucleated cells having f r o m t w o to t e n nuclei (Fig. 1D, E). A third observation was asynchronous
348
T. Benltez et al.: Instability of yeast heterokaryons
Table 2. Pedigree analysis during the first three divisions of 60 zygotes from the cross X51-1A (PI) x X51-1D (P2) Genotype of the clones
Buds
P1 heteroplasmons P2 heteroplasmons P1 * P2 heterokaryons P1/P2 synkaryons Non viable
Table 4. Pedigree analysis during the first division of 152 zygotes and the first division of H 1 buds, from the cross X51-2B (P2) x X51-2C (P1)
Zygote
H1
H2
H3
16 24 8 2 10
14 27 4 2 13
10 18 6 2 24
16 16 12 2 14
Genotype of the clones
Zygotes
H 1 bud
HI_ 1 bud
P1 heteroplasmons P2 heteroplasmons P1 * P2 heterokaryons P1/P2 synkaryons Non viable
50 35 27 5 35
70 37 13 5 27
75 45 8 5 19
152
152
152
Total
Table 3. Pedigree analysis of 21 fully viable zygotes and their buds from the cross X51-1A (PI) x X51-1D (P2) Type of zygotes
Number of zygotes
Type of buds H1
H2
H3
2 2 1 1 1
P2 P1 * P2 P1 * P2 PI*P2 P2
P2 P2 P2 P1 P1
P2 PI P1 * P2 PI *P2 PI
3 1 1 1
P2 P2 P1 * P2 P1
P2 P2 P2 P1 * P2
P1 * P2 P2 P2 P1
P2
2 1 1 1 1
P2 PI * P2 P1 P1 P1
P2 P2 P2 P1 * P2 PI
P2 P2 P2 P2 P2
Total
6
P1/P2
2
PI/P2
P1/P2
PI/P2
Total
21
PI *P2
Total P1
Total
divisions so that inside the same cell, one nucleus was dividing while the other was not (Fig. 1F).
Heterokaryon instability detected by pedigree analysis 1. Zygotes and their first three buds. In these experiments the strains X51-1A and X51-1D listed in the Table 1 were used.
a) Pedigree analysis on SD: In this experiment only H1 (central buds) were micromanipulated; 10 out of 110 zygotes analyzed were able to grow on SD. Of these, 5 were synkaryons and 5 heterokalyons. This frequency of synkaryons coincides with that expected from the leanness of the k a r l - mutation (Conde and Fink 1976). b) Pedigree analysis on YPD: H1, Hz and H3 buds were micromanipulated from 60 zygotes; H1 were always central buds; H2 and H 3 emerged from the same lateral side in 17 cases; from opposite lateral sides in 24 cases and one of them was central in 4 cases. The results obtained after analysing the colonies grown on YPD are shown in Table 2. There is a very high proportion of P1 and P2 heteroplasmons as compared to the number of heterokaryons. In the heteroplasmonic zygotes the number of P1 heteroplasmons is equal to that of P2 heteroplasmons, while in the first three buds H 1, H2 and H 3 this number is significantly higher for P2 heteroplasmons. This result indicates that unequal transmission of the nuclei to the buds is taking place. The viability of the micromanipulated buds decreases in such a way that H3 is less viable than He and this less than H1. The frequency of synkaryons observed in Table 2 does not differ from what was expected. There are two cases in which all the buds H1, H2 and H 3 are synkaryons. In these two situations the zygotes are also synkaryons. Table 3 shows the frequencies of different pedigree types for those cases in the previous experiment where both the zygote and the three zygotic buds, H1, H2 and H3, were fully viable. As in Table 2, the number ofheteroplasmonic P1 zygotes is equal to that of P2 zygotes, while in the buds the number of P2 heteroplasmons is higher than that of PI type. Unequal nuclear transmission is reinforced when one compares the ratio PI * P2 versus P1 + P2 of zygotic clones to that of the three zygotic buds (Table 3): 7 (Pl * P2) versus 12 (P1 orP2) for the zygotes, and 13
T. Benltez et al.: Instability of yeast heterokaryons
349
Table 5. Pedigree analysis of 100 fully viable zygotes, their first bud (HI) and the first bud of H 1 from the cross X51-2B (P2) x
X51-2C (P1) A Type of Zygotes
B Number of zygotes
P1 * P2
13 9 2 1
Total
25
P1
34 5 3 3
Total
45
P2
14 7 2 1 1
Total
25
P1/P2
5
Total
100
Type of buds
Type of buds H 1
H1
HI.1
P1 P2 P1 *P2 P2
P1 P2 PI*P2 P1
P1 P2 P1 * P2 P1 * P2
P2 P1 P1 * P2 P1 * P2 P1
P1/P2
P1 P2 P1 * P2 P1
P2 P1 P2 P1 * P2 P2
P1/P2
P1 * P2
Number of buds H 1
3 2 6
Total
11
P1
54 1
Total
55
P2
28 1
Total
29
P1/P2
5
Total
100
Type of buds H 1-1
P1 P2 P1 *P2
P1
P2
P2 P1
P1/P2
Data are independently organized according to the type of zygote A and to the type of H 1 bud B
(P1 * P2) versus 44 (P1 or P2) for the buds. These ratios are significantly different at the 95% level.
2. Zygotes, their first bud (111) and the first bud of H1 (1t1.1). We wanted to check if heterokaryon instability was due to a phenomenon occurring mainly in the zygotes. Such being the case, one can predict that, once the two nuclei have been transferred to the daughter cell, the probability of their being maintained through consecutive divisions should increase. To test this we have done a pedigree analysis in 189 zygotes. In these experiments the strains X51-2B and X51-2C listed in Table 1 were use:l. Zygotes, HI and H H buds were micromanipulated on YPD as described in Methods. The results are shown in Table 4. Only those zygotes from which it was feasible to separate the H I bud and from this the HI.1 bud are shown (152 zygotes). The proportion of P1 and P2 heteroplasmons is even higher than that of the previous experiment (Table 2). Also,
the relative numbers of P1 and P2 heteroplasmons indicate that the nuclei are not transferred with the same frequency. Frequencies of nuclear fusion (synkaryons) are comparable to previous results (Table 2). Table 5 shows the frequency with which heteroplasmons of each type, heterokaryons or synkaryons, are segregated when the zygotes and their buds H1 and Ha.1 are fully viable (100 zygotes). In this Table, as in Table 4, the unequal nuclear transmission is very accentuated (45 PI zygotes versus 25 P2 zygotes; 55 P1, H1 buds versus 29 P2, H1 buds; 58 P1, Hi-1 buds versus 31 P2, H1-1 buds). Out of 100 zygotes there are 34 and 14 colonies where the zygotes as well as the H 1 and the H1.1 buds are respectively P1 or P2, while the other nucleus has disappeared. There is also a fairly high frequency of inviable zygotes (Table 4). Unequal nuclear transmission is indicated by the 2 5 - 7 0 ratio of P1 * P2 versusPl + P2 in zygote colonies compared to the 11-84 ratio in H 1 colonies.
350
T. Benltez et al. : Instability of yeast heterokaryons
Table 6. Percentage of heterokaryon colonies obtained from the crosses described Mating strains a
Mating strains a
4A A10701C A941B SL107-8B 4836-8C MC40 5464-7C
JC25
ABQ21
14 15 14 20 13 8 10
9 12 12 12 20 4 16
percentage o f heterokaryons was even lower than in normal crosses. The results of heteroplasmon segregations did not differ between both types of crosses (data n o t shown). The effect o f isogenicity upon heterokaryon stability was also studied b y analyzing clones derived from crosses between isogenic strains. In spite o f an increasing degree o f isogenicity (up to 97%) stable heterokaryons were not obtained in any o f the crosses studied (data n o t shown).
Discussion
The results are the average of three experiments
1. Missegregation and nuclear inactivation as causes of heterokaryon instability Missegregation and nuclear inactivation in heterokaryon colonies: effect of the genetic background Table 6 shows the percentage o f heterokaryon colonies obtained in the different crosses. These percentages are n o t significantly different except in the crosses involving MC40, an a/a diploid strain. Table 7 shows the distribution o f synkaryons, heterokaryons and heteroplasmons, in the heterokaryon colonies analyzed from each cross. Auxotrophies, resistances or any other differences in the genetic backgrounds do n o t seem to affect the distribution o f the different genotypes, except in the cross with the strain MC40, the only diploid strain tested, which shows a very strong polarity in favor o f the diploid parental nucleus. The effect o f heterozygosis for mating type upon heterokaryon stability was studied. When crosses between strains o f the same mating type were tested, the
Our results indicate that heterokaryons are extremely unstable entities. The nuclei are not equally transmited from the mother to the daughter cells, due to two phenomena, missegregation and nuclear inactivation. The results that support missegregation are the following: a) Cytological observations (Fig. 1C). b) The frequency o f cases in Table 5 where zygotes are heterokaryons and H 1 buds heteroplasmons (22 lineages) compared with those where zygotes are heteroplasmons and HI buds heterokaryons (10 lineages). These frequencies should be similar if instability were only due to nuclear inactivation, c) The proportion of P1 and P2 heteroplasmons in the zygotes compared with that in the H1, H2 or H 3 buds (Table 2). Were nuclear inactivation the only reason for the appearance of heteroplasmons, the ratio should be the same in the zygote as in the H1, H2 and H 3 buds, which is not the case.
Table 7. Number of heterokaryon, heteroplasmon, and diploid clones obtained from heterokaryon colonies Mating strains
Mating strains (P1) JC25
ABQ21
(P2)
Heterokaryon P1 * P2
Synkaryons P1/P2
Heteroplasmon P1
P2
MC40 4A SL107-8B 4836-8C A10701C A941B
795 980 208 604 282 178
57 228 19 163 10 20
290 401 363 848 676 208
957 670 160 1,200 880 324
Total
1,820 1,302 531 3,316 1,028 641
Heterokaryon P1 * P2
Synkaryons P1/P2
208 138 328 478 55 667
44 13 7 30 0 75
Heteroplasmon P1
P2
140 156 565 104 120 1,896
448 307 424 278 130 1,962
Total
928 503 881 652 177 4,041
The heterokaryon colonies are mixtures of heteroplasmons, heterokaryons and synkaryons. Since a single colony may have heteroplasmons of each type, as well as heterokaryons and synkaryons, the total number of colonies is not the addition of the previous four colums. Data given are the results of three experiments. 9 independent colonies were analyzed in each experiment
T. Benitez et al.: Instability of yeast heterokaryons The existence of nuclear inactivation is supported by the following results: a) The high frequency of pedigrees in which a mother cell is heteroplasmonic while the corresponding daughter cell is heterokaryotic (Table 3 and 5). This cannot be explained only by missegregation, b) The existence of pedigrees with only one type of nucleus while the other nucleus has disappeared completely. In Table 3, the zygotes as well as H x , H2 and H 3 buds are all P2, in 2 cases. In Table 5, this happens in 34 cases for the P1 nucleus and 14 cases for the P2 nucleus, c) The high percentage of inviable zygotes, 25% in Table 2 and 23% in Table 4. d) Those cases in which both mother and daughter cells are heteroplasmons, but one of them is P1 while the other is P2 (Tables 3 and 5). e) Those cases in which there is a strong polarity in favor of one of the nuclei when this nucleus is 2n (Table 7).
351 Scheme 1
Zygotes
tll buds
HI_I buds
P1 or P2
P1 or P2
82 85%//t
/ Prior to first mitosis:
PI*P2
After first mitosis:
PI*P2 25
/
15% , PI*P2
95 l
7 54% / I
13 26%
46% ,
PI*P2 6
185% PI*P2 11
2. Differences in stability between heterokaryotic zygotes and heterokaryotic vegetative cells
3. Influence of the genetic background
Heterokaryon instability is present both in zygotes and in vegetative cells, although it is stronger in the former. The high frequency of heteroplasmons, which segregate even in the first zygotic bud (Table 2), together with the low frequency of heterokaryotic colonies (about 1% of the zygotes) and the size of these colonies, very small when compared to synkaryon colonies, led to the expectation of a frequency of heterokaryons among vegetative cells lower than it actually was. This means taht a strong selection against heterokaryons could already take place in the zygote, prior to or during the formation of the first zygotic bud. Once the first bud had both nuclei, the probability that they would be maintained in the cells seemed to increase. This could account for the fairly high fraction of heterokaryotic cells inside heterokaryon colonies (Fig. 1A and Table 7). To determine such differences in stability between heterokaryotic zygotes and vegetative cells we have compared the frequency with which a heterokaryotic cell is able to produce another heterokaryotic cell. In Table 5 it can be seen that only 3 out of 25 heterokaryotic zygotes produce heterokaryotic buds, whereas 11 heterokaryotic HI cells produce 6 heterokaryotic ones. Besides, from 100 viable zygotes from which the H 1 and H1-1 buds were also fully viable, we obtained the percentages of heterokaryons and heteroplasmons (shown in scheme 1), the frequency of transmission for both nuclei is 15% from the zygote to the first bud while the percentage is increased to 46% from this cell to its subsequent bud. Also, 26% of the zygotes continue to be heterokaryon after producing their first bud (H1). This frequency is 85% for the H 1 cell after producing its first subsequent bud. We therefore conclude that heterokaryosis is more stable in vegetative cells than it is in zygotes.
Another point to be discussed is the fortuitousness of the unequal transmission of the nuclei, as far as the genetic background (auxotrophies, drug resistances, sexual locus, isogenicity) is concerned. In Neurospora, homosexuality is an essential condition for obtaining stable heterokaryons (Gamjobst and Wilson 1956). We investigated if this was also the case for Saccharomyces, but our results did not support the hypothesis. Moreover, the number of heterokaryons obtained was lower than that obtained in heterosexual crosses. Nuclear inactivation could be due to a lack of identity in alleles of incompatible genes, as in Physarum (Carlile 1976). To test this hypothesis, strains of near isogenicity were constructed by means of five consecutive backcrosses (96,87% isogenicity). Nevertheless, heterokaryons were as unstable as those of non4sogenic strains. It is noteworthy that nuclear transmission is strongly polarized when a diploid nucleus is involved, and that it is the diploid nucleus the one which is preferentially transmited. Dutcher and Hartwell (1981, 1983a, b) have demonstrated that some gene products function both in nuclear division and in nuclear fusion, so that the lesion producing the defect in karyogamy is probably identical to the lesion producing the defect in cell division. If abortive mitosis occurs and some chromosomes are lost (Dutcher 1981), one should expect nuclear inactivation more frequently in haploid nuclei, because when a diploid nucleus is involved, the loss of a single chromosome would not lead to lethality, but only to an~uploid formation. The high viability of MC40 nuclei found in our results, as well as the very strong polarity in only this cross, support this point. Missegregation could occur because some type of clock controls-the bud emergence event, maintaining
352 only a single bud every cell cycle, and DNA synthesis and nuclear division can occur in the absence of bud emergence (Byers and Goetsch 1975). The nucleus in close proximity to the site of budding will migrate, producing a heteroplasmon, while the other one will divide, generally giving rise to a cell with three nuclei. This will explain the polynucleated cells found in vegetative cultures and zygotes (Figs. 1D and 1E). We have detected synkaryotic buds. only in the cases where the zygote was already a synkaryon. This means that nuclear fusion is a very precise event in the sexual cycle and secondary nuclear fusion hardly ever takes place. We conclude from our work that heterokaryon instability is a consequence of the structural and cell cycle limitations of the yeast cell, and then could hardly be improved.
T. Benitez et al.: Instability of yeast heterokaryons References Byers B, Goetsch L (1975) J Bacteriol 124:511-523 Carlile MJ (1976) J Gen Microbio193:371-376 Conde J, Fink GR (1976) Proc Natl Acad Sci USA 75:36513655 Dutcher SK (1981) Mol Cell Biol 1 : 245-253 Dutcher SK, Hartwell LH (1982) Genetics 100:175-184 Dutcher SK, HartweU LH (1983a) Mol Cell Biol 3 : 1255-1265 Dutcher SK, Hartwell LH (1983b) Cell 33:203-210 Garnjobst L, Wilson JF (1956) Proc Natl Acad Sci USA 42:613618 Hartwell LH, Culotti J, Reid B (1970) Proc Natl Acad Sci USA 66:352-359 Leonard TJ, Dick S, Gaber RF (1979) Genetics 88:13-26 Leonard TJ, Gaber RF, Dick S (1980) Genetics 89:685-693 Polaina J, Conde J (1982) Mol Gen Genet 186:253-258 Sherman F, Fink GR, Lawrence CW (1979) Methods in yeast genetics. Laboratory Manual. Cold Spring Harbor Laboratory, New York
Acknowledgements. We thank M. I. Carretero, A. Ferm[ndez and D. Su~rez for their skillful assistance, and E. Cerd~i, J. Polaina, I. L. Calder6n and K. Grohmann for helpful discussion. Financial support came for the "Comisi6n Asesora para la Investigaci6n Cientifica y Tecnol6gica" of the Spanish Government.
Communicated by B. S. Cox Received January 22, 1984
