Macronuclear chromatin changes during encystment in the ciliate Colpoda inflata: Formation of crystal-like structures in the resting cyst chromatin and nucleolar condensation

Europ. J. Protistol. 37, 121–136 (2001) © Urban & Fischer Verlag http://www.urbanfischer.de/journals/ejp

Macronuclear chromatin changes during encystment in the ciliate Colpoda inflata: Formation of crystal-like structures in the resting cyst chromatin and nucleolar condensation Ana Martín-González, Gemma Palacios and Juan Carlos Gutiérrez* Departamento de Microbiología-III, Facultad de Biología, Universidad Complutense (UCM), 28040 Madrid, Spain. Fax: # 91-394 49 64, E-mail: [email protected] Received: 26 June 2000. Accepted: 13 November 2000

We analysed the macronuclear chromatin structure changes during encystment of the ciliate Colpoda inflata, using both standard transmission electron microscopy and a spreading chromatin method, from isolated vegetative and resting cyst macronuclei. The vegetative macronucleus of C. inflata, like the majority of ciliates, is formed by discrete condensed chromatin clumps, which became fused during encystment. In mature resting cysts the macronuclear chromatin was similar to vegetative or resting cyst micronuclear chromatin probably indicating the transcriptional inactivity of the macronucleus in this stage. Likewise, the numerous nucleolar masses of the vegetative stage fused in only one nucleolar body during resting cyst formation, and in this nucleolar mass neither a granular zone nor nucleolar organizers were observed. Hybridization with a ribosomal DNA probe, after pulsed field gel electrophoresis of vegetative and mature resting cysts, showed that ribosomal DNA molecules could be compacted with some nucleolar elements, corroborating the nucleolar inactivation inferred from the ultrastructural data. The chromatin spreading method has revealed that vegetative macronuclear chromatin is formed by a net of long fibres organized in polynemic cords probably representing amplified regions of the macronuclear genome. It represents, as in the colpodid ciliate Bursaria, a type of non-classical polyteny. In this study we report for the first time in the genus Colpoda, a very special structural organization of the macronuclear chromatin in mature resting cysts: the existence of crystal-like hexagonal bodies. We also discuss widely all these macronuclear chromatin modifications and the factors that could be involved in the formation of polygonal structures. Key words: Chromatin crystal-like structures; Colpoda inflata; Encystment; Macronuclear chromatin; Resting cysts.

Introduction Vegetative cells of many species of ciliated protozoa can differentiate into resting cysts, under unfavourable environmental conditions like starvation [9, 20]. This differentiation process is designated encystment and involves progressive morphological and physiological changes concluding with *corresponding author

the formation of resting cysts [19, 20]. Among these changes, we can remark the following: a drastic decrease in cellular volume (in Colpoda inflata this volume loss is about 60%) [36], the formation of partially permeable barriers (cyst walls) which are composed of distinct cyst wall layers derived from different precursors [25, 38, 41, 66], organelle clustering as a consequence of cytoplasmic dehy0932-4739/01/37/02-121 $ 15.00/0

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dration [23, 41, 65, 66], a high autophagic activity [39] and drastic nuclear changes [19, 20]. Nuclear dualism (two types of nuclei simultaneously in one cell) is a general feature of ciliates. The micronucleus (Mi) is the germline nucleus while the macronucleus (Ma) is the somatic nucleus that determines the phenotype of the cell. Changes in both nuclei have been reported during ciliate encystment (for review see [21]). Some of the most important changes include: macronuclear chromatin condensation, with or without fusion of macronuclear masses [17, 18, 23, 24, 41, 42, 64-67], loss of macronuclear (Ma) DNA by forming extrusion bodies is sometimes detected in several ciliates [11, 16, 30, 36, 44], Ma DNA methylation pattern changes have been reported in C. inflata encystment [46] and an extensive reorganization of the Ma chromatin fine structure has been observed in some cases [56, 58, 62]. Probably, some of these changes may be involved in the control of gene expression, mainly for reaching the transcriptionally inactive state of the cystic macronuclear chromatin. Therefore, this microbial eukaryotic differentiation process must be highly controlled at both transcriptional and DNA conformational levels [22]. In the present study, we have analysed the macronuclear chromatin changes during encystment of the ciliate Colpoda inflata, using both: standard transmission electron microscopy and a spreading chromatin method, from isolated vegetative and resting cyst macronuclei. Likewise, pulsed field electrophoresis (CHEF) was applied to elucide the macronuclear rearrangement of the subchromosomal molecules in the resting cysts. This paper presents for the first time a very special structural organization (polygonal chromatin forms) in the genus Colpoda, and it is the second time that it had been reported in other colpodid ciliates [56]. Also, this is the first study on nucleolar reorganization and changes in ribosomal subchromosomic DNA during encystment in this group of ciliates.

Materials and methods Culture conditions and encystment induction Strain HSL-1 of Colpoda inflata was kindly supplied by Dr. E. Simon (University of Illinois, Urbana-Champaign, USA). This ciliate was monoxenically cultured at

31 ºC in C0.25E1 medium [36]. Encystment was induced by starvation [37].

Confocal microscopy (CM) Exponentially growing vegetative cells were fixed for 30 min with 70% ethanol at 4 ºC. After centrifugation (1,000 × g for 2 min) , the cells were resuspended in buffer 10 mM Tris / HCl, pH 6.8 with RNase (4 mg/ml) and incubated for 24 h at 37 ºC. After this, propidium iodide (PI) (15 mg /ml) was added to the cell suspension. Mature resting cysts were treated by the same way, excepting a previous treatment with lysis buffer [26] and easily sonicated in this buffer. Cell samples were examined using a Zeiss Axiovert 135 microscope with a confocal MRC 1,000 system (BioRad).

Standard transmission electron microscopy (TEM) Samples from vegetative and precystic cells at different times [13, 20, 25, 30, 36 and 40 h] from encystment induction, and mature resting cysts (two months old) were fixed for 30 min with 2% glutaraldehyde in PBS [phosphate buffered saline, pH 7.4). Then they were washed for 30 min in three changes of buffer and postfixed for 1 h in 1% buffered osmium tetroxide solution in PBS. After dehydration in increasing concentrations of ethanol, the samples were embedded in Spurr´s resin [60]. Ultrathin sections were double-stained with uranyl acetate and lead citrate and examined by using a Phillips EM-300 transmission electron microscope at 75 KV.

Chromatin spreading method For chromatin spreading we have used an adaptation of the Miller chromatin spreading technique [63]. Grids were coated with a thin carbon film (15–30 nm) [63, 69]. Colpoda inflata cell lysates were prepared by applying different methods depending on the cellular stage (vegetative, precystic or resting cyst). Vegetative cells were collected from the supernatant of a centrifuged exponential growing population maintained at 4 ºC during 5 min. From this point two different protocols were applied: a) vegetative cells were washed in nuclear isolation medium (5:1) [63]. Cellular lysis and chromatin spread preparation were carried out in drops of distilled water (adjusted to pH 9 with 0.1 mM sodium borate) on siliconized slides at 4 ºC for 20 min. b) vegetative cells were lysed in lysis buffer [26] containing 0.5% Triton X100, and macronuclei were isolated from this lysed cell solution by centrifugation. Precystic cells and mature resting cysts were disrupted using a glass homogenizer in lysis buffer with 0.5% Triton X100. Not disrupted cells and cyst walls were re-

Macronuclear chromatin changes during encystment of Colpoda inflata

moved by centrifugation, and the macronuclei from the supernatant were washed with nuclear isolation medium (5:1). Chromatin spread preparation was realized as for vegetative cells, but this protocol was applied during 20 min, 2 h or 6 h. From this point the protocol was the same for all cellular stages: The dispersed chromatin was transferred into microcentrifugation chambers and cold centrifugated (4 ºC) through a sucrose gradient (0.1 M) containing formaldehyde (1%) at 2,000 xg for 15 min. After centrifugation, the grids were removed from the chambers and immersed into a 0.4% Photoflo solution [Photoflo 200, Kodak) during 30 sec and air dried. Dehydration and staining was realized immersing the grids into a 1% phosphotungstic acid solution (pH 2) in 70% ethanol for 1 min. For complete dehydration the grids were subsequently immersed in 70% ethanol (30 sec) and then in 100% ethanol (30 sec). Grids were dried in isopenthanol (30 sec). Rotary shadowing of grids was realized with Pt-Pd wire at an angle of 8º. Finally, the samples were examined by using a Zeiss transmission electron microscope at 80 KV.

Pulsed-field gel electrophoresis and hybridization Preparation of samples for pulsed-field gel electrophoresis was carried out according to the protocol previously described [40]. The pulsed-field electrophoresis system was a contourclamped homogeneous electric field (CHEF) with a running time of

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40 h (20 h with a switching interval of 20 sec and 20 h with a switching interval of 40 sec). Saccharomyces cerevisiae S13 chromosomes were used as size standards. The ribosomal probe used was a fragment (555 bp) obtained from the 18S ribosomal gene of C. inflata. This probe was obtained and 11-dUTP labeled with digoxigenin by PCR as described [40]. Gel denaturation and Southern blotting to Nylon + were carried out according to the methodology described [54]. Hybridizations were performed as described in the manual for the Dig Luminescence Detection Kit from BoehringerMannheim.

Results The confocal microscopic study of nuclei of both types of differentiated stages (vegetative cell and mature resting cyst) has revealed great differences (Fig. 1). The vegetative Ma (macronucleus) of C. inflata showed chromatin distributed in condensed regions of very variable sizes, which were included into a karyoplasmic region constituted by diffuse chromatin and including regions without doublestrand (ds) DNA (without PI) (Fig. 1 A). Besides, the regions of condensed chromatin were organized by small chromatinic bodies of variable fluorescence intensity. The vegetative Ma had a larger diameter of 11.6 µm and a minor one of 9.1 µm.

Fig. 1. Confocal micrographs of macronuclear system of both vegetative cell (A) and resting cyst (B) of Colpoda inflata, after staining with propidium iodide. Drastic differences about the macronuclear chromatin condensation of both macronuclei can be observed. In 1B the ellipsoidal micronucleus is observed (arrow). Bar indicates 5 µm.

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The mature resting cyst Ma (Fig. 1 B) showed a PI fluorescence very homogeneous throughout the nuclear body, no diffuse chromatin or regions without ds DNA were detected. The resting cyst Ma had a larger diameter of 8.6 µm and a minor one of 6.3 µm approximately. In the same picture (Fig. 1 B), an ellipsoidal micronucleus (Mi) with, likewise, a homogeneous fluorescence was observed. We calculated the approximate volume of both vegetative and cystic Ma, by using its larger and minor diameters; the vegetative Ma presented a volume of about 492.2 µm3, while the cystic macronuclear volume was around 169.4 µm3. Therefore, we estimated that the macronuclear volume loss during encystment of C. inflata was about 65% with respect to the vegetative Ma volume.

Ultrastructural study by TEM According to the previously studied encystment kinetics of C. inflata [4], we had selected some precystic phases to study the nuclear ultrastructural changes of this ciliate. In vegetative cells the Ma chromatin was organised by many discrete chromatin lumps which were included into a fibrillar matrix (Fig. 2). The vegetative Ma also presented many nucleolar masses in clusters with a homogeneous aspect and filling a large region of the Ma. In some of these nucleolar masses an associated nucleolus-organizing region could be observed (Fig. 2). The vegetative Mi was ellipsoidal and presented a continous network of chromatin strands (Fig. 2). Generally, in vegetative cells, the Mi was located in a Ma cavity without nuclear membrane fusion between both nuclear systems. After encystment induction, in early precystic cells (20 h after induction), the Mi was separated from the Ma and the nucleolar masses became fused together generating larger and more irregular nucleolar masses (Fig. 3). Also, the small bodies of the Ma chromatin were slightly larger at this encystment time. The successive fusion of nucleolar masses during the first encystment phases of C. inflata became more remarkable in late precystic phases, as is shown in Fig. 4, in which a precystic Ma (30 h after induction) with three large nucleolar masses was observed. Between 25 and 36 h of encystment kinetics a macronuclear extrusion took place, of about 1/3 of the Ma volume. In Fig. 5, a 36 h old precystic

cell with a quite elaborated cyst wall was observed; it presents a chromatinic region separated from the Ma (arrow in Fig. 5), which could be an extrusion body. In Fig. 6, another 36 h precystic cell shows a lenticular Mi, that was also the characteristic micronuclear form in mature resting cysts, and, again, it appeared near the Ma surface. In this precystic phase only one large nucleolar mass was detected, indicating that all single nucleoli had been fused to only one mass. Likewise, this precystic stage showed a quite dehydrated cytoplasm with organelles and membranous material clustering. Finally, Fig. 7 shows the nuclear system of a mature resting cyst [two months old). The condensed chromatin of both nuclei was very similar and a high cytoplamic dehydration with mitochondrial clustering, autophagosomic residues and abundant membranous material was observed.

Study of isolated macronuclear chromatin After nuclear isolation of vegetative, precystic cells and mature resting cysts by applying one of the methods indicated in Materials and Methods, a chromatin spreading technique was applied. In Fig. 8 A, a general view of a complete vegetative Ma, incubated in 0.1 mM borate buffer (pH 9) during 20 min, is showed. The spread chromatin originating from the macronuclear mass consisted of a net of long fibres organized in polynemic cords (Fig. 8 B), formed by numerous fibres 50–70 nm thick. Sometimes, the vegetative chromatin of C. inflata presented polygonal bodies of very variable sizes, generally with six sides (hexagonal) (Fig. 9). In the same micrograph, we can appreciate two forms of spread chromatin fibres: bundles of linear fibres which diverged from different points of the polynemic cords and bundles with fibres scattered as festoons (arrow in Fig. 9). After applying the same protocol to mature resting cysts of C. inflata (two months old), we observed that the majority of the Ma chromatin was formed by condensed polygonal chromatin (hexagonal bodies or lamellae) (Fig. 10). These bodies were similar to those observed in vegetative cells, but, contrarily, they were more numerous and basically formed the complete macronuclear chromatin of the resting cyst. The size of these polygonal bodies was very variable, with an estimated area on the average of about 7.7 µm2. Most

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Fig. 2. Transmission electron micrograph of vegetative nuclear system of C. inflata. Ma: macronucleus, Mi: micronucleus (×19,121). Fig. 3. Electron micrograph of a 20 h precystic macronucleus. The nucleolar masses fuse producing larger nucleolar masses (×20,625). Fig. 4. A precystic Ma (30 h after encystment induction) with three large nucleolar masses (arrows) (×27,500). Fig. 5. A 36 h old precystic cell, showing a chromatinic region (arrow) separated of the Ma, which could be a very early extrusion body (×14,850).

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Fig. 6. Nuclear system of the 36 h precystic cell that shows a lenticular Mi and only one large nucleolar mass (arrow) into the Ma (×24,750). Fig. 7. Mature resting cyst nuclear system of C. inflata (two months old). Ma: macronucleus, Mi: micronucleus (×28,500).

of these polygonal lamellae were hexagonal with unequal sides (Fig. 10 A). These chromatinic hexagons were formed by compacted nucleosomal fibres (Fig. 10 B). Therefore, these polygonal structures represented a very special supranucleosomal organization originating from the regular aggregation of macronuclear nucleosomal fibres. Upon incubation in 0.1 mM borate buffer during 2 h instead of 20 min, the chromatin of the resting cyst Ma presented the same polygonal configuration without any decompactation (data not shown). A partial decondensation of these polygonal structures was obtained after incubation in 0.1 mM borate buffer overnight. After this treatment, some spread fibres from the polygonal aggregate were observed (data not shown). The same spreading protocol was applied to precystic cells. In Fig. 11, a sample of Ma chromatin from precystic cells, 84 h old, after standard incubation with borate buffer, is shown. Small irregular aggregates of chromatin that could be the core for

the polygonal body formation were detected in these cells (Fig. 11).

Pulsed-field electrophoresis analysis of the vegetative and resting cyst macronuclear DNA and location of rDNA Due to macronuclear polyploidy, there is much more Ma DNA than Mi DNA in the cell, so the lysed-cell preparation was effectively Ma DNA. The pulsed field electrophoresis resulted in a heterogeneous population of bands for both vegetative cells and resting cysts of C. inflata (Fig. 12 A), ranging between 90 and 2,000 Kb in size. By densitometric scanning and integration of the peaks, a mean size of 1,045 Kb was obtained from the vegetative Ma DNA molecules of C. inflata. The electrophoretic pattern of subchromosomic molecules from resting cyst Ma was basically similar to the vegetative pattern (Fig. 12 A, lanes 3 and 4).

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Fig. 8. Chromatin spreading technique. A: A general view of a complete vegetative Ma, which is formed by a net of long fibres organised in polynemic cords (arrow) (×26,000). B: An amplification of the area into the box of figure 8 A (negative version) showing macronuclear fibres organized in polynemic cords (×100,000). Fig. 9. Vegetative chromatin of C. inflata. Two forms of spreaded chromatin fibres and polygonal bodies are observed (×31,250).

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Fig. 10. Resting cyst chromatin of C. inflata: Hexagonal bodies or lamellae are detected (A) (×28,000). B: An amplified view of these bodies, which are formed by compacted nucleosomal fibres (×56,000).

Fig. 11. Precystic macronuclear chromatin 84 h old, after standard incubation with borate buffer. Small irregular aggregates of chromatin are observed (arrows) (×31, 250).

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Fig. 12. A: Pulsed field gel electrophoresis of vegetative cells and resting cysts of C. inflata. Lanes 1 and 2: Vegetative cells, lanes 3 and 4: Resting cysts, lane M: DNA size standards (S. cerevisiae S13 chromosomes). B: Chemilumigraph (digoxigenin detection method, after hybridization with the ribosomal probe). Lanes 1 and 2: Vegetative cells, lanes 3 and 4: Resting cysts, lane M: DNA size standards. Arrow indicates the hybridization positive band in vegetative cells. (*): Positive hybridization into the wells in lanes 3 and 4.

Although the electrophoretic pattern was almost a continuous population of bands, we could differentiate two well defined Ma populations of molecules. The first one was located in the upper electrophoretic resolution limit of the gel, at the same level of the higher chromosomic band (about 2,000 Kb in size) of S. cerevisiae. In this region we could distinguish two or three conspicuous bands clustered in about 0.5 cm of gel. The second molecular region of this pattern was included between the third size marker (about 1,100 Kb) and more ahead of the last size marker (260 Kb). This region (5.5 cm of gel) was formed by a continuous population of bands in which it was sometimes possible to distinguish some discontinuities or discrete bands. Two thick bands were distinguished from the rest of the pattern, 700 and 200 Kb in size, respectively. These two bands were lost in the second region of resting cyst electrophoretic pattern.

The location of the rDNA in the macronuclear genome of C. inflata was performed using a PCR originated fragment (555 bp in size) from the 18S ribosomal gene of this ciliate as probe. Hybridization with this rDNA probe showed that the 18S ribosomal gene was located as a single band in the native Ma DNA pattern of vegetative cells. Only one hybridization positive band of about 200 Kb in size was detected (arrow in Fig. 12 B). This subchromosomic band corresponded to one of the more conspicuous bands detected in the second region of the electrophoretic pattern. Likewise, this probe showed a positive heterologous hybridization with chromosome XII of Saccharomyces cerevisiae (2,000 Kb) (lane M in Fig. 12 B), in which the ribosomal genes are located [6]. However, in mature resting cyst samples (lanes 3 and 4 in Fig. 12 B) no rDNA positive band in the electrophoretic pattern was detected, only a positive hybridization in the wells (agarose blocks with lysed cells) was observed (* in Fig. 12 B).

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Discussion Vegetative and resting cyst macronuclear chromatin analysis From the confocal microscopy study of vegetative Colpoda inflata we conclude that the vegetative Ma chromatin is composed of condensed regions that are included within a karyoplasm with diffuse chromatin. This general view of the Ma agrees with that observed using other light microscope staining protocols such as Feulgen [36] or DAPI (data not shown). Besides, the most condensed regions are composed of others with very variable fluorescence intensity, which could correspond to the so called “small bodies” that are observed by TEM. On the other hand, the Ma chromatin of mature resting cysts presents a homogeneous fluorescence with PI all over the macronuclear mass, and no “empty” fluorescence regions corresponding to the fibrillar diffuse not condensed chromatin, are detected. The approximate loss of Ma volume during encystment has been calculated and is about 65%. This value is proportional to the estimated cytoplasmic volume loss of 65% [37]. A similar relation between the cytoplasmic and Ma volume loss has been also found in the hypotrich ciliate Gastrostyla steinii [18]. If this correlation is observed in other encysting ciliates too, it might be generalized that during ciliate encystment the Ma volume loss is proportional to the cytoplasmic volume loss, in order to maintain a constant nucleo-cytoplasmic ratio. In this nuclear volume reduction both, chromatin condensation and macronuclear DNA loss can be involved. The standard electron microscopy (TEM) has shown that the vegetative Ma of C. inflata is formed by discrete condensed chromatin lumps, the socalled “small bodies” [51], in agreement with the confocal observations. These “small bodies” are present in the majority of ciliates [51, 52], they are 0.1–0.2 µm in size with an irregular form, representing condensed chromatin fibres. These bodies are interconnected by filaments, which are better recognized when isolated chromatin is examined [52]. Therefore, we can state that the vegetative chromatin of C. inflata presents an organization very common among ciliates, such as: several from the Kinetofragminophorea class (Didinium, Coleps, Lacrymaria, Tillina, Colpoda, Bursaria and Balantidium) or the Oligohymenophorea (Tetrahymena

and Paramecium) and also the Polyhymenophorea (Spirostomum, Stentor and Euplotes) [51, 52]. The vegetative Mi of C. inflata is, likewise, a typical colpodid Mi, ellipsoidal in shape and with condesed chromatin, and, as in other ciliates, the Mi is located in a macronuclear cleavage [51]. In early precystic cells (20 h after encystment induction) some significant changes are recognized in the Ma: an increase in size of the chromatinic “small bodies” is reported, indicating the beginning of Ma chromatin condensation by fusion of these bodies. Between 25 and 36 h after encystment induction, the precystic cells of C. inflata show a still incomplete cyst wall, and the somatic cilia are reabsorbed [36]. This precystic phase is corresponding to the so-called “red spherical cell with an orange halo” stage, according to the study on the encystment kinetics of this ciliate [4]. During this encystment period a macronuclear extrusion takes place, which results in a chromatin loss of about 1/3 of Ma volume. This extrusion body contains both chromatinic and nucleolar materials. The extrusion process does not eliminate exclusively rDNA, as it was suggested by Frenkel [15] with respect to the extrusion body of Colpoda steinii, but it is a rather unspecific process that eliminates any macronuclear region: only chromatin or both chromatin and nucleolar elements. Biochemical and autoradiographic data have shown that the DNA of extrusion body does not differ from the bulk Ma DNA [51] and, consequently, it can not be exclusively a specific DNA, such as rDNA. The formation of extrusion bodies or chromatin extrusion is a very common phenomenon among ciliates. Generally it occurs during Ma division, but it may also occur during clonal ageing in Paramecium [51] and during encystment in some ciliates like colpodids. Both, division and encystment extrusion bodies have been reported in colpodids, such as: Colpoda maupasi, C. inflata [36], C. cucullus [30, 44], C. steinii [15], Tillina magna [3, 16), T. canalifera, Bresslaua sicaria, B. vorax and B. insidiatix [5]. However, in other colpodids, such as: Colpoda aspera [5] and Cyrtholophosis elongata [14] this phenomenon does not exist. Precystic cells of C. inflata (36 h old and later) present a lenticular Mi, as it was previously described by Martín-González et al. [36] using optical microscopy, and the Mi chromatin does not show any modification with respect to the vegeta-

Macronuclear chromatin changes during encystment of Colpoda inflata

tive cell, as it has been reported in other colpodids [3, 5, 10, 44] and hypotrichs [42]. The Ma chromatin of this phase, like in mature resting cysts, is similar to vegetative or resting cyst Mi chromatin, which may indicate the gene inactivity of the Ma in this phase and later. The application of the chromatin spreading method to vegetative Ma chromatin analysis of C. inflata revealed that this chromatin is mainly formed by a net of long fibres organized in polynemic cords, which are supranucleosomal in nature. Probably they represent amplified regions of the Ma genome. This pattern of branching of chromatin fibrils probably represents an oligoteny phenomenon, perhaps it is only present in some regions of the Ma genome of this ciliate, or perhaps it is a generalized phenomenon. Similar structures to those found in C. inflata have been described exclusively in the colpodid Bursaria ovata [57] and also it has been supposed in another species of the same genus: B. truncatella [55]. Sergejeva and Bobyleva [57] considere that this type of polyteny should be considered as hidden local polyteny, since it can only be detected by electron microscopy. Therefore, it cannot be considered a classical polyteny like the giant chromosomes of the hypotrich Ma anlagen [51]. So, the authors suggest that the vegetative Ma of Bursaria presents another type of non-classical polyteny perhaps specific for polyploid differentiated somatic nuclei of some ciliates, but we think that this idea should be confirmed by studying more species of ciliates. In fact, our results on C. inflata are a good confirmation of those observations on B. ovata [57]. As it was described by transmission electron microscopy of ultrathin sections, the Ma chromatin of C. inflata, like the majority of ciliates, is formed by the so called “small bodies”, which are interwoven by chromatin fibres. In B. trucatella [58, 62] and Didinium nasutum [27], using the chromatin spreading method, these “small bodies” have been identified as dense chromatin clumps 100 to 200 nm in size, and they represent transcriptionally inactive chromatin. Upon incubation in 0.1 mM borate buffer these clumps decompact forming loop-shaped chromatin fibres [62], and the decompact degree is proportional to the incubation period. In Didinium nasutum [27] a transition pattern between the chromatin clumps and the nucleosomal chains (active chromatin) has been observed. It is formed by inactive chromatin clumps of 19–25 nm in size and, likewise, they can

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be decompacted by borate buffer incubation. We have not observed these Ma chromatin clumps in vegetative cells of C. inflata, probably because they were completely decompacted by the borate buffer incubation. Conjunctly to the polynemic fibres of C. inflata vegetative cells, a low number of chromatinic polygonal structures, generally hexagonal, appear. These hexagonal bodies are formed by compacted nucleosomal fibres and they represent the main Ma chromatin structure in the resting cysts of this ciliate. In the resting cysts of C. inflata these hexagonal structures are very plentiful and they can be partially decompacted by overnight incubation with borate buffer, releasing nucleosomal fibres and showing that these bodies are formed by the same Ma chromatin nucleosomal fibres as vegetative cells. In 84 h old precystic cells of C. inflata, which correspond to the last precystic phase of the encystment kinetics [4], aggregation bodies of fibres, that may be considered as an intermediate structure of the characteristic definitive polygonal bodies in mature resting cysts, have been observed. These irregular chromatin aggregations could be the initial nucleation centers to form these polygonal structures. During encystment of B. truncatella [58], peculiar chromatin aggregations (named chromonemes) were described, and some of these types of structures are similar to those irregular chromatin aggregations observed in precystic cells of C. inflata. Likewise, all these structures, after complete decompactation, are constituted of nucleosomal fibres. For the first time, these resting cyst hexagonal chromatin bodies were observed in two ciliated species of the genus Bursaria [B. truncatella and B. ovata) [56]. These authors differentiate two structural types of hexagonal lamellae in the Ma chromatin of precystic and mature resting cysts of both species: type-I formed by superhelical 15 nm thick threads twisting into a spiral structure, and type-II with a lot of short thread fragments (2–4 nm thick) and minicircles and/or minirosettes. Polygonal structures formed by spiral chromatin fibres also have been observed in resting cysts of C. inflata. The finding of similar chromatin structures in C. inflata confirm, in the first place, the results reported twelve years ago in two species of Bursaria [56] and in the second place, it is the second time that these special structures have been observed in

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colpodid ciliates, being also the first time that it has been detected in the genus Colpoda. At present, these chromatin structures have been only described in these two ciliated genera, they are present in little amount in vegetative cells and they form all Ma chromatin in mature resting cysts. The possibility that these structures might be artefacts originated by the technique is voided by the two following reasons. The real nature of these structures is exhibited after borate buffer incubation (they decompact in nucleosomal fibres), and a recent autoradiographic electron microscopy analysis [59] in B. ovata has shown that 3H-dthymidine is incorporated into these polygonal chromatin structures indicating the presence of DNA in them. We do not know how these polygonal structures are formed or what is the real mechanism involved in the formation of these structures, but we can consider some factors which could be involved in their formation, such as the biophysic of concentrated DNA solutions. An important factor to be considered is the high level of dehydration that the vegetative cell undergoes during encystment, which induces an increase of cytoplasmic density, organelle clustering and Ma chromatin condensation. In addition to the effect of specialized basic proteins in DNA condensation, the spontaneous ordering of DNA into liquid crystalline phases that occurs at high concentration has been taken into account as a leading mechanism in chromatin packaging [33, 34]. Both, bacterial nucleoid and eukaryotic nuclei are ordered in fluid macromolecular crowded media, where the DNA concentration is locally in the range of 50–500 mg/ml [34] and highest DNA concentrations (800 mg/ml) can be still found in some living systems such as in the head of bacteriophage T4 [31]. In the same range of concentration DNA forms in vitro liquid crystalline phases that are also highly ordered structures. Two types of phases can be distinguished, which depend on the polymer concentration: “cholesteric” type and “columnar hexagonal” phase [for higher DNA concentrations). In vivo similar organizations are found: the cholesteric DNA organization has been described in dinoflagellate chromosomes [35] and in some bacterial nucleoids. Hexagonal packing of DNA molecules was found in bacteriophages and sperm nuclei [33]. The formation of this liquid crystalline chromatin organization reported in several organisms is basi-

cally due to two factors: condensation (a high ratio DNA concentration / volume) and macromolecular dehydration. In ciliate encystment both of these factors exist, therefore the polygonal crystal-like Ma structures observed in resting cysts of Bursaria ovata, B. truncatella [56] and Colpoda inflata could be formed by these same factors. Recently (68), a prokaryotic DNA protection by stress-induced (starvation) biocrystallization has been reported in Escherichia coli. A co-crystallization of the DNA with the stress induced protein Dps is considered in this study as a generic defense strategy againts stress conditions. From all this we think that these Ma crystal-like hexagonal structures only found until now in colpodid ciliates, are specific chromatin structures of the Ma of these ciliates, which are almost exclusively forming Ma chromatin in the resting cysts. Furthermore, it is very usual to find crystal-like or paracrystal forms in both cytoplasm and Ma of encysted ciliates [13, 23]. They are formed, likewise, by the dehydration that the cell undergoes during encystment.

Nucleolar changes during encystment C. inflata has a composite nucleolus, which is formed by numerous nucleolar masses distributed throughout the Ma. These nucleolar masses are not fused like in C. steinii [15, 51], but rather they are distributed in apparently uncoupled clusters and surrounded by the chromatinic “small bodies”. From a structural view point, the nucleolar masses of C. inflata are very similar to those in Tetrahymena [51]: a fibrillar packed cupped zone, that consists of preribosomal RNA bound to proteins and a granular zone with ribonucleoprotein granules (the precursors of ribosome subunits). Also, as in Tetrahymena and other colpodids (C. maupasi, C. steinii and C. simulans) [51], C. inflata presents a nucleolar chromatin body in the depression of the cupped nucleolar mass, which is the nucleolar organizer (rDNA). Likewise, some nucleolar masses are located near the Ma envelope, as in Tetrahymena, which is in contrast to a central composite nucleolus observed in some colpodids [51]. In precystic cells (20 h old), the nucleolar masses of the vegetative composite nucleolus are beginning the fusion, generating larger and irregular nucleolar masses. The most drastic changes observed in the nucleolar system are produced in later precystic

Macronuclear chromatin changes during encystment of Colpoda inflata

cells (30 h after induction), in which only two or three big nucleolar masses are detected. These fused nucleolar masses present the fibrillar zone, while the granular zone becomes undistinguishable or is almost exclusively reduced to an internal region surrounded by the fibrillar zone. Likewise, the nucleolar organizers are not detected. These nucleolar changes are probably indicating intensive biosynthetic modifications at ribosomic level, such as a gradual decrease of ribosome production during encystment process. It is known that the nucleolar morphology can undergo drastic changes depending on the physiological stage of the eukaryotic cell [43]. In ciliates, a similar phenomenon has been reported during some peculiar situations, e.g.: in Paramecium [51] its multiple nucleoli fuse when the ciliate is undergoing starvation, and during the stationary phase of culture growth the small nucleoli of Tetrahymena fuse in bigger aggregates [51, 52]. A similar phenomenon occurs in Tetrahymena under the action of RNA synthesis inhibitor Actinomycin D, cadmium ions or ultraviolet irradiation [51, 52]. We suggest that these phenomena and encystment are related, because starvation is the most universal inducer of ciliate encystment and the stationary phase appears when food becomes deficient. Also, during starvation conditions the biosynthetic reactions like RNA synthesis, decrease [20]. This could explain the parallelism among these phenomena (starvation, blocking biosynthetic reactions) and the ciliate encystment process, in which these characteristics are also involved. Nucleolar changes during encystment have also been reported in other ciliates. In C. cucullus [29], during early phases of encystment, the nucleolar spherical bodies fuse. Chessa et al. [12] report that in stationary phase cells of C. cucullus, precystic and mature resting cysts, the nucleolus becomes fragmented in several aggregates. In Tillina magna [16], one hour after encystment induction, the nucleolar fibrillar material increases in size, which is explained as a nucleolar fusion. The granular region is smaller in precystic cells 3 hours old, but the number of these nucleolar granules increases in the, so named by the author, “early resting cysts”. In one week old resting cysts of Bursaria truncatella [49], a part of the nucleolar material undergoes segregation and it is extruded from the macronucleus to the cytoplasm in which it is eliminated. In precystic cells of C. inflata (36 hours old) and mature resting cysts, only one nucleolar mass is

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observed without a granular zone indicating the absence of new ribosome formation and protein biosynthesis. This high condensation of the biosynthetic ribosomal mechanism may be related with the results of rDNA hybridization in resting cysts as discussed below.

rDNA subchromosomal localization of vegetative and resting cysts by pulsed field gel electrophoresis Total DNA of both vegetative and resting cysts of C. inflata (basically Ma DNA due to Ma polyploidy) show an electrophoretic mobility in standard electrophoresis system (data not shown), very similar to those reported in Hymenostomatia, such as Tetrahymena thermophila and Glaucoma chattoni [28], in the heterotrich Blepharisma japonicum [53] and the Peniculina Paramecium [61]. Therefore, C. inflata presents a Ma organization composed by subchromosome-size DNA molecules [50, 52], which can only be resolved by pulsed field gel electrophoresis. The pulsed field gel electrophoresis analysis (CHEF) shows that the Ma genome of this ciliate is composed by a heterogeneous and almost continuous population of subchromosomic molecules, ranging between 90 and 2,000 Kb (average size of 1,045 Kb). A few ciliate genomes have been analyzed by using this method: OFAGE [orthogonal field alternation gel electrophoresis) has been applied to Tetrahymena thermophila [1], Paramecium tetraurelia [47] and P. primaurelia [8], and the CHEF modality has been applied to Blepharisma japonicum [2, 48], P. primaurelia [7] and T. thermophila [32]. By OFAGE the average size for T. thermophila Ma molecules was found to be about 1,100 Kb [1], while by CHEF the molecular size range obtained was between 100 and higher than 1,100 Kb [32]. In four different species of Tetrahymena [45] the average value was found between 1,000 and 1,100 Kb. Therefore, the Ma molecules of C. inflata like other species of Colpoda [40] present an average size around 1,000 Kb, which is more similar to the average size (1,100 Kb) of T. thermophila and it is different from the average sizes reported for other ciliates, such as: Paramecium with averages of 300 Kb [8] or 450 Kb [47], Glaucoma chattoni (average range of 100–300 Kb) [28] and Blepharisma japonicum [2] with 100 to 170 Kb. On the other hand, the pulsed field gel electrophoretic pattern of

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C. inflata vegetative cells is more similar to that of P. tetraurelia [47] and P. primaurelia [7], but quite different from Tetrahymena species [1, 45], in which a discontinuous and well definite population of bands form the complete pattern. Hybridization with the rDNA probe shows that the 18S ribosomal gene is located as a single band in the native Ma DNA pattern of C. inflata vegetative cells. This band (about 200 Kb in size) coincides with a thicker band of the pattern, indicating the differential amplification of these genes into the ciliate Ma genome [50]. Only in two cases ribosomal genes have been detected after applying pulsed field gel electrophoresis [both using OFAGE): T. thermophila [1] and P. tetraurelia [47]. The results obtained in C. inflata are more similar to those reported in T. thermophila (ribosomal subchromosomic positive band of 350 Kb), than those showed in P. tetraurelia with a ribosomal band higher than 500 Kb in size [47]. Recently, we have found [40] a karyotypic variability in rDNA subchromosome size among colpodid species, and these size variations in the rDNA subchromosomal molecule seem to be species specific. In fact, in some colpodid species like C. maupasi the ribosomal subchromosomic band size (450 Kb) is more near to P. tetraurelia band size (500 Kb) than that of T. thermophila. Although the electrophoretic pattern of mature resting cyst Ma molecules of C. inflata is not very different with regard to a vegetative one, hybridization results with the ribosomal probe were completely different. No positive band in the resting cyst Ma DNA electrophoretic pattern was detected, only positive hybridization was observed in the well agarose blocks. We have two interpretations for these results: a) Both, disrupted and undisrupted resting cysts are probably present in the agarose blocks of resting cyst samples. Therefore, undisrupted cysts and cyst walls could prevent the normal electrophoretic mobility of DNA molecules. However, we think that this interpretation is not valid, because the electrophoretic pattern of resting cyst Ma DNA is almost the same as the vegetative pattern, including subchromosomic bands bigger than ribosomal molecules. So, there is no apparent reason to obtain a negative hybridization in the resting cyst pattern. b) rDNA is located in nucleoli and according to the ultrastructural study during encystment of C. inflata, in which the nucleolar morphology undergoes drastic changes, it might be possible that rDNA molecules

could be compacted with other nucleolar elements. These elements could obstruct the release of these molecules from the agarose block to the resolving gel and so, positive hybridization is only obtained in the agarose block. At present, we do not know what is the mechanism and the mode by which these molecules could be compacted, and the nucleolar elements involved in this condensation. In the near future, a more extensive study on it should be carried out by testing (with the rDNA probe) different DNA precystic patterns. Acknowledgements: The authors wish to acknowledge Dr. M.T. Trendelenburg (Institute of Cell and Tumor Biology. German Cancer Research Center. Heidelberg. Germany) who provided to G.P. the possibility to apply the chromatin spreading method in C. inflata. This research work was supported by a grant from Dirección General de Investigación Científica y Técnica (DGICYT), project: PB96-0611 to J.C.G., and a predoctoral fellowship from Comunidad Autónoma de Madrid (CAM) to G.P.

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