The micronucleus of the ciliate Stylonychia mytilus; its nucleic acid synthesis and its function

Experimental

THE MICRONUCLEUS ITS NUCLEIC

Cell Research 61 (1970) 6-12

OF THE CILIATE

STYLONYCHIA

MYTILUS;

ACID SYNTHESIS AND ITS FUNCTION D. AMMERMANN

Zoologisches Institut der Universit& 74 Tiibingen, West Germany

SUMMARY The micronuclei of Stylonychia mytilus have a Gl phase of 8-9 h and an S phase of 2 h. No G2 was observed, and micronuclear mitosis starts before macronuclear DNA synthesis has ended. Experiments in which the micronuclei were removed from the organism showed that they have a function during vegetative growth and division. After removal of the micronuclei their function is taken over by the macronucleus. Sometimes amicronucleate animals form “pseudomicronuclei”. They contain less DNA than diploid micronuclei, but this fact is apparently compensated by their increased number. Their removal effects the same malformations that can be observed during the first few months after removal of the diploid micronuclei. This suggests that the pseudomicronuclei have a function probably like the function of the micronuclei. Autoradiographic exneriments showed that there is nrobably a low level of RNA-synthesis in the micronuclei during the whole cell life cycle.

In most ciliates we find two kinds of nuclei: macronuclei and micronuclei. The macronucleus controls the metabolism of the cell; its loss results in death. The micronuclei have an important function during conjugation. After the old macronucleus becomes pycnotic, one or more of the descendants of the micronuclei give rise to a new macronucleus. The function of the micronuclei during vegetative growth and division of the cells is, however, not clear. Although there are some species which are able to grow as well without as with micronuclei, there are other species-sometimes only other strains of the same species-which are not viable without micronuclei (for review see [16]). In addition RNA has been spectrophotometrically detected in the micronuclei of Paramecium caudatum [8], and RNA-synthesis has been autoradiographically demonstrated in micronuclei of Paramecium caudatum and Paramecium aurelia [9, 121. It is therefore clear that the micronuclei of at least some ciliates Exptl Cell Res 61

are actively engaged in the metabolism of the cell. Some ciliates are apparently intermediate. Amicronucleate clones are well known, but removal of the micronuclei in the laboratory causes malformations, a low division rate, and death of a high percentage of the clones. The best known examples are Tetrahymena pyriformis and Paramecium bursaria. Amicronucleate strains of T. pyriformis (see [3]) and P. bursaria [7] can be found in natural habitats, but removal of the micronuclei lowers the vitality and the division rate of P. bursaria [13], and causes death of most T. pyriformis clones [16]. Stylonychia mytilus belongs to this group. Amicronucleate clones have not yet been found in natural habitats. Frick [6] attempted unsuccessfully to obtain amicronucleate clones of S. mytilus using p-radiation from a strontium source. In the present study amicronucleate clones were successfully obtained using UV-microbeams and X-irradiation.

7

Micronucleus of the ciliate Stylonychia mytilus Therefore it seemed interesting to investigate the role of the micronucleus in the cell life cycle and the effect of its removal. In addition, RNA and DNA synthesis by micronuclei was studied.

METHODS The details of cultivation and the technique of crossing are described earlier [l]. For irradiation, two different methods were used: (1) Single animals were carefully compressed between a slide and a quartz cover glass until the micronuclei were visible. These were then irradiated with a UV-microbeam which had a diameter just slightly greater than the micronuclei. After several seconds’ irradiation the animals were freed and put in culture fluid with food. The radiation instrument was constructed from Dr Czihak [4]. (2) Several animals were collected in 0.3 ml culture fluid and irradiated with X-rays (330 kR) produced by the instrument “Mtiller RT 100”. The animals were then isolated and fed. The microspectrophotometric measurements were made with a UMSP I (Zeiss). The animals were fixed in alcohol/acetic acid (3: l), stained according to Feulgen and the DNA content was measured at a wavelength of 560 nm. To investigate DNA synthesis aH-thymidine (Radiochemical Centre, Amersham) was added to the culture fluid for a given time, then the animals were washed and olaced on a subbed slide. To studv RNA synthesis, animals were fed with Tetrahymenh labeled heavily with SH-uridine (Radiochemical Centre, Amersham). It was necessary to isolate the micronuclei with a microuipette after lysis of the cells with tritomspermidine (for details see [ll]). They were placed on a subbed slide and fixed with alcohol and acetic acid (3 : 1). Some of them were treated in the usual manner with enzymes and then prepared for autoradiography.

RESULTS The DNA synthesis of the micronuclei To find the time of DNA synthesis during the cell cycle, dividing cells from a clone with a generation time of 12 h (21°C) were isolated. Every 30 min 100 &i/ml 3H-thymidine was added to the culture fluid of a group of these cells. The animals were incubated for 30 min, then washed and prepared for autoradiography. The results are shown in fig. 1. Micronuclear DNA synthesis begins after the macronuclear DNA is half replicated and

Gl

s

D

MXi-ONICIWS Cl

S

D

Micronucleus

Fig. 1.

is completed before macronuclear replication is finished. Mitosis begins immediately after the completion of micronuclear DNA synthesis (fig. 2). There is no G2, neither in the macronucleus nor in the micronucleus. This cell cycle is different from that of Euplotes [lo], but similar to the cycle of Urostyla weissei [17]. The cell after removal of the micronuclei There is no difference with respect to the later development of the clones between the effects of UV- and X-irradiation. Thus there is no need to distinguish between these two irradiation methods in the following sections. In most cases X-rays were used because the procedure of irradiation is easier (see Methods). Of all clones of different origin that have been irradiated up to the present, 30% did not survive the treatment. The clones deriving from the irradiated cells died some days after irradiation. The majority of irradiated cells showed another reaction. Isolated animals seemed to develop normally during the first 3 to 4 divisions after irradiation, just like unirradiated controls. During the subsequent divisions, however, most of the descendants after a division were malformed cells. They were rounded, unable to move normally or to feed and finally died. These malformed cells, which contain a macronucleus, are described in detail by Frick [6]. Fig. 3 shows some examples. Exptl Cell Res 61

8

D. Ammermann

Fig. 2. Nuclei of an animal, labeled with 3H-thymidine 10 to 104 h after division. The macronuclei have not yet finished DNA synthesis (the replication bands have not reached the end of the macronucleus) while the micronuclei are enlarged and elongated and are beginning to divide. Feulgen, x 530. Fig. 3. S@on&ziu myiilus, malformed cells from a clone some days after X-ray irradiation. Phase contrast, x 450. Fig. 4. (a) Cell with diploid micronuclei; (6) cell with pseudomicronuclei (arrow). Feulgen, x 860. Fig. 5. Micronucleus of an animal, labeled 2 h with $H-uridine in the Gl period and treated with DNase. x 3300. Exptl Cell Res 61

Micronucleus of the ciliate Stylonychia mytilus

9

Table 1. Number of animals in clones from irradiated (330 kR) and unirradiated cells (only healthy animals counted) + clone died out Days Clone no.

1

2

3

5

7

9

I1

14

17

20

23

4 8 8 4 10 7 9 4 S 12 2 4 8 5 2

4 3 8 2

3 6 6 1

:i

::

40 8 8

40 6 2

30 20 20 6 2

30 70 100 3 25

30

80

100

150

:

6 I1 10

1 40

;b

50

120

: 4 2 3

: 2 I 3 10 1 6 7 1 2

4 20 10 1 4 1 2 1

8 12 16 1 4

I; 3 4 2 3

7 7 10 1 1 1 3 2 1 IO 4 5 3 5

10 10 2 10

15 10 1 10

50 20

60 15

70 50

200 100

5

15

30

50

8 5 16 10 9

32 28 40 31 35

160 150 200 180 250

Irradiated 1 2

: 5 6 s’ 9 10 11 12 13 14 15

Unirradiated ; 2 e

Sometimes only malformed cells arise after a division, resulting in the death of the clones. In most clones, however, some healthy animals survive. They are able to feed and to divide. Their descendants are either malformed cells or normal animals. During the subsequent 2 to 3 months the percentage of healthy animals in the clones increased, and the percentage of malformed cells decreased. Table 1 shows the development of 15 irradiated and 5 unirradiated clones. All clones were derived originally from one clone that was divided before irradiation. After 3 months the clones usually remain stable, i.e. they produce only few or no malformed cells, and the division rate is sometimes as high as in the unirradiated controls (under the best conditions one division in 12 h), sometimes lower. It should be emphasized that the slow growth of the clones (table 1) is not caused by a low division rate, but rather by mal-

formation and death of most of the descendants within a clone. During the first month after irradiation the number of malformed cells in a clone is always much higher than the number of healthy animals. A cytological investigation shows that all clones that develop in this manner have lost their micronuclei. The macronucleus is sometimes divided into three instead of two parts, but otherwise it shows no abnormalities. The reason for the abnormalities might either be irradiation-induced loss of the micronuclei, or else more general damage to the macronucleus and/or cytoplasm. Two experiments were performed in order to distinguish between these hypotheses: (a) Cytoplasmic regions near the micronucleus were irradiated with UV-microbeam. These regions were at the same distance from the macronucleus as were the micronuclei, and were irradiated in the same manner as in the experiments described earlier. The Exptl Cell Res 61

10 D. Ammermann clones derived from these animals developed like the unirradiated control cells, and all animals possessed micronuclei. (b) Stabilized clones without micronuclei (6 months after irradiation) were irradiated with 330 kR as described earlier. Six days after irradiation the average number of animals in clones that were derived from irradiated cells was 108 (n =40, G = +44), the average number in unirradiated control clones was 124 (n =23, u= k 34). No malformed cells could be found in the irradiated clones. From these data it is clear that the irradiation damages amicronucleate cells only slightly, if at all. These experiments show that the loss of the micronuclei is the reason for the described disturbances. The micronuclei of Stylonychia mytilus must therefore have a function during vegetative growth. Which cell organelles replace the micronuclei after their loss? After the loss of the micronuclei another part of the cell can apparently take over the micronuclear functions. To find out whether or not this is done by the macronucleus, the following experiment was carried out: animals with normal diploid micronuclei were crossed with stabilized healthy amicronu.cleate clones. The conjugating pairs and later the two exconjugants of a pair were isolated. Both exconjugants contain a hemicaryon derived from the haploid gamete nuclei of one of the partners, and the hemicaryon gives rise to haploid micronuclei and polyploid macronuclei. If, in the amicronucleate clones, the macronucleus has taken over the function of the micronucleus, one would expect that both exconjugants of a pair would become sensitive to irradiation, because the “‘adapted” macronucleus does not survive the conjugation. If, however, another part of the cell has taken over the function of Exprl

Cell Res 61

the micronuclei, one would expect that one exconjugant would survive irradiation without damage while the other would be damaged. The result of the experiment was that after irradiation all 20 exconjugant clones derived from 10 conjugation pairs showed the damage described earlier. This demonstrates that following removal of micronuclei their function is taken over by the macronucleus. Formation of “pseudomicronuclei” In the last 4 years the fate of 15 amicronucleate clones has been observed over a long period of time until their senescence. Cytological study after different times of cultivation showed that cells of four clones contained small micronucleus-like, Feulgen positive bodies near the macronucleus. Some weeks after their first appearance no amicronucleate animals could be found in these clones. The animals with “pseudomicronuclei” apparently grew faster than the others. These structures were almost certainly formed from the macronucleus. They look like micronuclei and behave similarly during mitosis. During conjugation, however, they cannot substitute for the micronuclei. Exconjugations from a cross “animals with a pseudoanimals” micronuclei” X “amicronucleate develop a tiny macronuclear anlage, but it disappears after a day, and no exconjugant ever survived (several hundred tested). It has not been possible to investigate the DNA content of all clones with pseudomicronuclei, but one of the clones was investigated in detail and compared with diploid clones. The pseudomicronuclei (fig. 4) are smaller (diameter 2 p) than the normal diploid ones (diameter 5-6 p). Spectrophotometric measurements showed that the pseudomicronuclei have only 21% of the DNA that is present in diploid micronuclei (pseudomicronucleus: 36 units, (T= + 11, n =22; diploid micro-

Micronucleus of the ciliate Stylonychia mytilus nucleus: 168 units, (T= + 30, yt = 23). The number of pseudomicronuclei per cell, however, is larger, i.e. 12.1 pseudomicronuclei per cell (a = i4.1, n = 100). The number of diploid micronuclei is different in different clones. In the clones that were investigated the average number was between 2 and 4 per cell. This shows that the clone with pseudomicronuclei has approximately the same content of “micronuclear-DNA” as the normal diploid cell. This result fits together with a result mentioned in another paper [l]. Animals with haploid micronuclei contain on the average twice as many micronuclei as animals with diploid micronuclei These results support the idea that the diploid and haploid micronuclei and the pseudomicronuclei have a function in the vegetative cell. To test further whether or not these pseudomicronuclei have a function during the vegetative growth of the cells, the clones were irradiated (330 kR) in the same manner as described earlier. Eight days after irradiation the animals in the clones were counted. The average number of healthy animals in clones deriving from irradiated cells was 12 (mean deviation c = i 8, n = 27) and in addition to these healthy animals numerous malformed cells were present. The mean number in un-irradiated clones of the same age was 167 (g = +47, n = 12). All descendants of irradiated cells had lost their pseudomicronuclei. From these data the conclusion can be drawn that the pseudomicronuclei have a function like the normal micronuclei, and that their removal causes the same damage to the cell as the removal of the normal micronuclei. Experiments with 3H-uridine The previous sections show that the micronuclei of S. mytilus have a function. The question arose whether this function is expressed in the synthesis of RNA. To answer

11

this question the incorporation of 3H-5Turidine into RNA was investigated. Dividers were isolated and, during different stages of the subsequent cell cycle, some of them were isolated and fed with Tetrahymena labeled heavily with 3H-uridine. Two hours later the micronuclei were isolated and prepared for autoradiography. After an exposure time of usually 2 months the micronuclei showed some label (fig. 5). RNase removed most of this label. There were no striking differences in the number of grains during the different stages of the cell life cycle. Micronuclei of all stages showed a RNase-sensitive label. From these results the conclusion could be drawn that the micronuclei synthesize RNA. However two major objections could be made: (a) The surface of the micronuclei could be contaminated with labeled cytoplasmatic particles. Stevens (unpublished, see 1121) investigated this problem and showed with electron microscopic methods that in Eupfotes there is no cytoplasmic contamination of nuclei isolated in tritomspermidine. But in S. mytilus the cytoplasmic label is so enormously much higher than the micronuclear label that a small amount of adherent cytoplasm could simulate label in the micronucleus. (The cytoplasm and the macronucleus are completely black in autoradiographs after some days exposure, while micronuclei show at least 20-50 grains after 2 months.) (b) If there is RNA in the micronuclei, it is not necessarily produced there, since it could have migrated into the nucleus. It was not possible to answer these objections experimentally and therefore the experiments do not show beyond doubt that there is RNA synthesis in the micronuclei. DISCUSSION The present observations on the role of the micronucleus in S. mytilus are comparable Exptl Cell Res 61

12 D. Ammermann with some earlier observations made by Schwartz [ 131 on Paramecium bursaria. He showed that the removal of the micronucleus in this species severely hampers viability. After several months the clones regain their former division rate. This shows that the micronucleus must have a function. In some amicronucleate clones new micronuclei are formed from the end of the macronuclei [ 141. They are, however, apparently not smaller than normal micronuclei. P. bursaria was the first species in which a new formation of micronuclei from macronuclei has been observed. It would be interesting to investigate the effect of the removal of these new micronuclei and to compare this with the corresponding results described in this paper. The experiments performed with &JIZonychia showed that after the removal of the micronuclei the macronucleus can take over its function(s). The macronucleus therefore does contain at least some genes normally active in the micronucleus. The question remains open why it takes such a long time to activate genes in the macronucleus after the loss of the micronucleus. Another remarkable fact is that sometimes the macronuclei form new pseudomicronuclei which then have micronuclear functions. Their removal causes the same damage to the cells as the removal of diploid micronuclei. From this the conclusion can be drawn that the adaption of the macronuclei in amicronucleate cells, i.e. the activation of special gene(s), is cancelled after formation of pseudomicronuclei. It is interesting to compare these results with results obtained from genetic experiments with Paramecium aurelia. Sonneborn [15] and Pasternak [9] constructed cells with different alleles in the macronucleus and the micronucleus. The micronuclear genes they tested could not contribute to the phenotype of the cell when the appropriate genes in the macronucleus were lacking. Whether these Exptl Cell Res 61

genes in the micronucleus cannot be activated or whether the amount of RNA they produce is too small for the large cell remains open. When serotype transformation is induced, the micronuclei show an increased rate of RNA synthesis [9]. Whether or not this function of the micronuclei is essential for the cell, is unknown. The results described in this paper show that the micronuclei have special functions in S. mytilus which cannot be taken over easily by the macronucleus. It seems that the relationship and the distribution of the functions between the macronucleus and the micronuclei is well balanced and more complex than supposed earlier. The author is indebted to Dr David M. Prescott for his support, and wishes to thank Miss Meister, Dr Phil., who made some of the measurements with the UMSP I. This work was supported by the Deutsche Forschunasnemeinschaft. A PaA of this investigation was made during my stay at the Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, Co10 80302, USA.

REFERENCES 1. Ammermann, D, Arch Prot 108 (1965) 109.

2. 3. 4. 5. 6. 7.

- Chromosoma 25 (1968) 107. Corliss, J 0, J protozool i (1954) 156. Czihak, G, Zeiss-Mitteilungen 2 (1961) 165. Dembowska, W, J exptl zoo1 43 (1926) 485. Frick, D, Biol Z 86 (1967) 629. Golikowa. M N., Propr (Abstracts .., in orotozool . of papers read at the 3rd Intern. congr. on protozoology, Leningrad). Publishing House Nauka, Leningrad (1969). 8. Moses, M J, J morph01 87 (1950) 493. 9. Pasternak, J, J exptl zoo1 165 (1967) 395. 10. Prescott. D M, Kimball. R F & Carrier. R F, J cell b&l 13 (1962) 175.. 11. Prescott, D M, Rao, M V N, Evenson, D P, Stone. G E & Thrasher. J D. Methods in cell physiol, vol. 2. Academic &ess,‘New York (1966). 12. Rao, M V N & Prescott, D M, J cell biol 33 (1967) 281. 13. Schwartz, V, Z Naturforsch 2b (1947) 369 14. - Biol Z 77 (1958) 347. 15. Sonneborn, T M, Microbial gen bull 11 (1954) 25. 16. Wells, C, J protozool 8 (1961) 284. 17 I . Jerkadziadosz, M & Frankel, J. In press. Received January 7, 1970