Nuclear changes, macronuclear chromatin reorganization and DNA modifications during ciliate encystment

Europ. J. Protista/. 34, 97-103 (1998) June 16, 1998

European Journal of

PROTISTOLOGY

Minireview

Nuclear Changes, Macronuclear Chromatin Reorganization and DNA Modifications during Ciliate Encystment Juan Carlos Gutierrez, Ana Martin-Gonzalez and Sergio Callejas Departmenta de Micrabialagia-III, Facultad de Biolagia, Universidad Camplutense, 28040 Madrid, Spain

Summary In this paper we review one of the most important aspects of ciliate encystment; the nuclear and chromatin changes involved in the differentiation process that forms the resting cyst or cryptobiotic stage. All these changes are directed to obtain the main feature of any cryptobiotic form: the gene-silencing and the genome preservation. The nuclear changes include: macronuclear fusion and chromatin condensation, formation of chromatin crystal-like structures in some ciliates, nucleolar fusion and rONA inactivation, macronuclear DNA loss and specific DNA modifications.

Key words: Nuclear changes; Chromatin reorganization; Ciliate encystment.

nuclear changes [28]. Here we will consider only one aspect of ciliate encystment, the nuclear changes. Nuclear dualism is a general feature of ciliates and changes in both kinds of nuclei have been reported during ciliate encystment. These changes include: macronuclear (Ma) chromatin condensation, Ma-DNA loss, changes in the Ma-DNA methylation pattern, nucleolar changes and an extensive rearrangement of the Ma chromatin. All these modifications probably serve to interfere with gene expression, and are indicative of transcriptional inactivity, the main characteristic of any cryptobiotic form.

Macronuclear Fusion and/or Chromatin Condensation Introduction Vegetative cells of many species of ciliates can differentiate into resting cysts under unfavourable environmental conditions, such as starvation [13, 28]. This differentiation process, encystment, constitutes a true cryptobiosis phenomenon according to Keilin's definition [34]. Excystment is the process of emergence from the cryptobiotic stage (resting cyst). Together these processes form the E-E (encystment-excystment) cycle, with two stable differentiated states; the vegetative and the cystic or cryptobiotic one [28]. Ciliate encystment involves progressive and drastic morphological and physiological changes, including a drastic decrease of cellular volume (in hypotrichs this volume loss is 70-80% and in colpodid ciliates is 60-70%) [43], the presence of partially permeable barriers (cyst walls) which are composed of distinct cyst wall layers derived from different precursors [30, 45, 48, 70], organelles clustering as a consequence of cytoplasmic dehydration [27, 47, 69], a high autophagic activity [46] and drastic © 1998 by Gustav Fischer Verlag

In general, during encystment, macronuclei in ciliates with several macronuclei (Ma) in the vegetative stage fuse to form only one cystic macronuclear (Ma) mass. This process has been reported mainly in stichotrichs and hypotrichs ciliates, such as Gonostomum sp. [69], Stylonychia mytilus [72], S. pustulata [33], Laurentiella acuminata [29], Onychodromus acuminatus [31], Gastrostyla steinii [26], Oxytricha hi/aria [33,66], O. /allax [25], O. nova [8], Histriculus muscorum [49] and Sterkiella histriomuscorum [1]. An exception to this rule has been observed in the stichotrich Pleurotricha sp. [47] and Urostyla grandis which have many small Ma in vegetative cells and resting cysts [60]. In the last species, several Ma degenerate during encystment, so the resting cyst has a lower number of Ma than the vegetative cell [65]. Macronuclear fusion causes a drastic volume reduction and chromatin condensation. In those ciliates with only one Ma mass, there is a Ma volume reduction and chromatin condensation, for instance: Euplotes rariseta [15], E. taylori [24], Diophrys

98

J. C. Gutierrez, A. Martin-Gonzalez and S. Callejas

scutum [70], Colpoda cucullus [32, 36], C. steinii [21], C. inf/ata [43], Til/ina magna [22], Bursaria truncatel/a [64] and B. ovata [62]. Apparently, this Ma fusion and/or condensation is similar to the condensation that takes place in preparation for division during the G z period in ciliates with multiple Ma, or during physiological reorganization or posttraumatic regeneration of ciliates [57]. All these instances of Ma fusion are correlated with some cortical changes, e.g. buccal reorganization, cortical division and/or total or partial kinetosome reabsorbtion during encystment. Some authors suggest that Ma chromatin is redistributed and that the configuration of chromatin elements changes when the Ma condenses [57]. It could favour the homogeneous repartition of genetic material between both daughter cells during division. Besides a Ma reorganization, Ma fusion during encystment could have another meaning: to maintain constant the nucleocytoplasmic ratio in the cell. A high correlation between the cytoplasmic and Ma volume loss has been found in some ciliates [26, 44]. If this correlation is observed in other encysting ciliates, it suggests that, during ciliate encystment the Ma volume loss and cytoplasmic volume loss occur in parallel, perhaps as a consequence of dehydration, so that a constant nucleocytoplasmic ratio is maintained. Curiously, Urostyla grandis, an exception among stichotrich ciliates without Ma fusion during encystment [60], shows Ma fusion of its over 100 Ma during cell division [56]. In this case, the nucleo-cytoplasmic ratio could be maintained by degeneration of several Ma during encystment [65]. How does Ma fusion occur? At present, we know very little about the elements involved in the Ma fusion and how they work. In both division and encystment, microtubules are present during Ma fusion. High numbers of microtubules seem to be involved in several macronuclear changes during stichotrich cortical morphogenesis, including: Ma fusion by enlargement of interconnecting isthmuses, mixing of the Ma chromatin during condensation and elongation, and Ma division [68]. During encystment a high microtubular density in both Ma and/or micronucleus (Mi) has also been described in Gastrostyla steinii [73], Histriculus muscorum [49], Stylonychia mytilus [72], Til/ina magna [22] and Telotrochidium henneguyi [71]. There are several hypotheses for the function of microtubules during encystment [73]: Microtubules may function passively by providing a rigid karyoskeletal guide for the fusing Ma or they may actively widen the isthmus joining the Ma. Likewise, the microtubules, via their insertions into the nuclear envelope and chromatin, could provide the motive force responsible for the chromatin condensation [73]. This last hypothesis has been used to explain the presence of microtubules in Ma during encystment [49,

71,72]. However, Ma chromatin condensation can apparently be realized in the absence of microtubules [1, 25, 27, 31, 66, 69]. Therefore, other factors like cell dehydration as well as microtubules may be involved in the encystment Ma fusion and/or condensation. The presence of Ma microtubules has also been reported during excystment [31, 73], they may have an important function during the excystment Ma amitotic division that occurs in some ciliates. Macronuclei are the main nuclear elements undergoing condensation during encystment because micronuclear (Mi) chromatin condensation has been only reported in few ciliates [37, 72]. Macronuclear chromatin condensation is accompanied by drastic changes in chromatin organization and ultrastructure, e.g., in stichotrich ciliates, Oxytricha fal/ax [25], Histriculus muscorum [49], Laurenttella acuminata [27], Gastrostyla steinii [73], Stylonychia mytilus [72], Pleurotricha sp. [47] and O. hifaria [66], large spheroidical bodies are usual Ma chromatinic structures in the resting cysts. In general, an increase in size of Ma chromatin bodies is reported during encystment [15, 20, 32, 55], which is due to the fusion of smaller chromatin bodies. This compactation is specially high in some colpodid resting cysts, Bursaria truncatella [63], B. ovata [62] and Colpoda inf/ata [55]. In these species, crystal-like hexagonal chromatin structures (liquid crystal type) have been observed in the Ma chromatin of resting cysts, after the application of chromatin spreading procedures. Probably, the high level of dehydration that the cell undergoes during encystment is an important factor in the formation of these polygonal structures. Spontaneous DNA ordering into liquid crystalline phases at high concentration has been hypothesized as an important mechanism in chromatin packaging [39]. These liquid crystaline phases, which depend on the polymer concentration, can be found in vitro and in vivo, e.g., hexagonal packing of DNA molecules (columnar hexagonal phase) was found in bacteriophages and sperm nuclei [38]. The formation of this liquid crystalline chromatin organization, reported in several organisms, is presumably due to both, condensation and macromolecular dehydration. These Ma chromatin crystalline hexagonal structures, only found until now in colpodid ciliates, may be a resting cyst specific form of chromatin packing. Besides, crystal-like or paracrystaline bodies are commonly found in both cytoplasm and macronucleus of encysted ciliates [14, 27], which are formed by the cell dehydration. In addition to these factors involved in the chromatin packing, we must also consider the effect of specialized basic proteins. At present, there is only one study on this topic using Gastrostyla steinii [26], in which a micro spectrophotometric analysis reveals that the cystic Ma had about 1.23-fold more histones than a

Ciliate nuclear modifications and encystment single vegetative Ma mass and that these cystic Ma histones were about 1.59-fold more arginine-rich than the vegetative macronuclear ones. This may be suggesting that the high Ma DNA condensation in resting cysts could be due to the presence of arginine-rich proteins (such as protamines), as in metazoan cells where substitution of protamines for histones in spermatogenesis is correlated with an extremely dense chromatin packing and loss of RNA synthetic capacity. However, a study of nucleosomes spacing in the chromatin of Oxytricha fallax [67] has shown that the vegetative spacing with a repeat length of 198 bp is maintained in the resting cyst. The Ma chromatin condensation during encystment may be involved in the transcriptional inactivation of the resting stage. Another example of molecular DNA aggregation is probably shown in the resting cysts of Oxytricha sp. [2], in which the electrophoretic pattern of the genesized Ma DNA molecules (0.4-20 Kb in vegetative cells) is almost completely restrained to one big electrophoretic band (>20 Kb size) in the resting cyst Ma DNA. This molecular aggregation could be similar to the in vitro aggregation of the DNA molecules reported in Stylonychia mytilus [41], obtained by incubating the Ma DNA under increasingly stronger ionic conditions, because the electrophoretic patterns look very similar. Other nuclear changes involved in ciliate encystment include: nuclear envelope changes, modification of the pattern of nuclear pores, micronuclear degradation and autogamy. Changes to both, nuclear envelope and/or nucleopores have been shortly studied. During encystment in Euplotes rariseta [15] there are drastic changes in nuclear pore number and distribution pattern. The hexagonal pattern of vegetative nucleopores changes to a lineal distribution in the resting cyst and their number is considerably reduced. During excystment the vegetative number of nucleopores is recovered, as shown in Onychodromus acuminatus [31]. This reduction in the number of Ma pores may indicate a decrease of biosynthetic capacity in the resting cyst. Micronuclei with numerous envelopes or membranous layers in the resting form have been reported in several ciliates [27, 37, 72, 73]. This could represent protection against the high autophagosomic activity during encystment. In fact, a Mi number reduction, due to Mi degradation during encystment, has been reported in some ciliates, e.g. Laurentiella acuminata [29], Gastrostyla steinii [73] and Oxytricha fallax [25]. The vegetative average Mi number is restored by mitosis during excystment [25,29], and a Mi DNA synthesis previous to mitosis has been observed [29]. In these species the excystment Mi mitosis is coincident with the amitotic Ma division that restores the average number of vegetative Ma masses.

99

Sexual processes (autogamy) have been only reported during encystment in Tetrahymena rostrata [12,26]. The encystment of this ciliate can be divided in two phases, a first pre-autogamic phase in which the cell forms the cyst wall from mucocyst secretion [50] and a second phase in which the autogamy takes place. From a phylogenetic view point, this system has a double advantage; a complete regeneration of the old Ma by autogamy, which decreases cell ageing [12], and a resistance mechanism against unfavourable environmental conditions (resting cyst).

Nucleoli and Encystment In general, nucleolar structure changes take place in the cell as response to many external factors and as a consequence of the stage in the cell cycle [58]. Besides, this nuclear organelle may be an indicator of the cellular biosynthetic level. Drastic changes in Ma nucleoli occur during encystment, which indicates important changes in the rRNA metabolism. Three main types of nucleolar modifications can be distinguished during ciliate encystment: structural modifications with nucleolar fusion (Colpoda cucullus [32], C. inf/ata f55], Tillina magna [22] and Dileptus visscheri [37]), structural modifications without fusion (Stylonychia mytilus [72], Gastrostyla steinii [73], Oxytricha fallax [25] and Histriculus muscorum [49], all of them stichotrich ciliates) and nucleolar disappearance (Sterkiella histriomuscorum [1] and Kahliella simplex [20]. In Pleurotricha sp. [47] and Telotrochidium henneguyi [17] any nucleolar alteration has been reported. Nucleolar morphology undergoes drastic changes depending on physiological state of the cell [51]. For example, in Paramecium [57] and in Tetrahymena [57, 58] the multiple nucleoli fuse under starvation or stationary phase conditions. Similar changes occurs in Tetrahymena under the action of RNA synthesis inhibitor Actinomycin D, cadmium ions or ultraviolet irradiation [58]. We may find a short parallelism among these phenomena (starvation, blocking biosynthetic reactions) and the encystment process [28]. Ciliate encystment depends on both RNA and protein synthesis [28], suggesting that the Ma is transcriptionally active at least in the early encystment stages. Some authors [10, 22] think that the increase of the granular nucleolar component observed in the precystic cells of some ciliates may be explained by the necessity for rRNA synthesis for the cyst wall molecular biosynthesis. In late precystic phases the biosynthetic activity decreases and so the rRNA requirement would be lower, which may explain the drastic ultrastructural changes that nucleoli undergo during encystment, such as disappearance or condensation. Probably, nucleolar

100

J. C. Gutierrez, A. Martin-Gonzalez and S. Callejas

changes are connected with the Ma chromatin changes resulting in a transcriptionally inactive resting cyst Ma. In mature resting cysts of C. inf/ata [55], only one nucleolar mass without a granular zone is observed, indicating the absence of new ribosome formation and protein biosynthesis. This high condensation of the biosynthetic ribosomal mechanism may be related with the rDNA sub chromosomal location in the resting cyst pulsed field gel electrophoretic pattern [55], and this molecular observation might confirm the nucleolar inactivation in resting cysts by condensation or packing.

Macronuclear Extrusion Bodies Macronuclear extrusion occurs in both, division and encystment. It may take place during the Ma division of Tetrahymena, Colpidium [57], Urocentrum turbo, Ancistruma isseli, Allosphaerium convexa and others [6], or after Ma division, such as in division cysts of many colpodida [6, 21, 36, 43]. Like in cell division, a Ma chromatin extrusion occurs during encystment of many colpodid ciliates [3, 6, 22, 36, 43, 53], but not in others as Colpoda aspera [6] and Cyrtolophosis elongata [16]. In general, Ma extrusion involves the formation of only one extrusion body, but in some cases more than one extrusion body is formed during encystment, e.g. Colpoda maupasi undergoes double extrusion during the cold induced encystment [53], and in Tillina magna, the extrusion occurs repeatedly during the encystment process [22]. Chromatin extrusion is not very common in stichotrich and/or hypotrich ciliates, but has been reported in Pleurotricha lanceolata during encystment [42]. The amount of Ma DNA loss during extrusion depends on vegetative Ma DNA amount, as it was reported in Colpoda cucullus [52]. Therefore, the amount of lost Ma DNA is very variable among cells of a heterogeneous encysting population. A flow cytometry DNA study in C. inf/ata (unpublished data) has shown that about 47% of the Ma DNA is lost during encystment. This amount represents the average Ma DNA amount of an extrusion body. What type of Ma DNA is lost? Is Ma extrusion an unspecific or specific process? The few data available on these questions indicate that the extrusion process does not eliminate exclusively rDNA, as it was proposed by Fenkel (1980) [21], rather it is an unspecific process that eliminates any Ma region with only chromatin or both chromatin and nucleolar material, as observed by electron microscopy in Colpoda inf/ata (unpublished data). Biochemical and autoradiographic data have shown that the DNA extrusion body does not differ from the bulk Ma DNA [57], and, conse-

quently, it can not be considered a specific DNA, such asrDNA. At present, we do not know how this process is discharged and how it is regulated. Several hypotheses concerning to explain the physiological significance of this DNA loss during both division and encystment have been presented. In 1930, Calkins [7] explained it as a "purification" process and Kidder (1933) [35] named it "cleaning" macronuclear process. Kidder and Claff (1938) [36] considered that the DNA reorganization involved in the extrusion body formation replaced the absence of conjugation in these ciliates. Faure-Fremiet (1953) [18] supposes that this extrusion process is a regulation mechanism of macronuclear polyploidy, by eliminating the "extra" genomes or maintaining the chromosomic macronuclear equilibrium by extrusion of "extra" chromosomes. In Tetrahymena thermophila [11] the DNA content of the division extrusion bodies is not a multiple of the Mi DNA content, so the chromatin extrusion can not be considered the elimination of whole genomes from Ma. According to another hypothesis, chromatin extrusion is a means to regulate the nucleocytoplasmic ratio [57]. After division and encystment, the cell volume is reduced and as the nucleocytoplasmic ratio must be maintained, the nuclear volume must be also reduced, and it can be obtained by chromatin condensation and/or chromatin extrusion. We think that perhaps this last assumption is the most appropriate, taking into account the strong correlation between both cytoplasmic and nuclear volume reductions during encystment. Besides a Ma DNA reorganization is involved during this extrusion process. A Ma DNA loss without extrusion body formation could be also occur during encystment. In fact, in Oxytricha sp. [40] a reduction of the total Ma DNA to about the half of the average content has been reported. However, we must be circumspect when we explain data from cytophotometry or microspectrophotometry because, as Gutierrez (1985) [26] noted, a "theoretical loss" of Ma DNA corresponding to a loss of absorbancy may be due to the high chromatinic condensation, which presents a greater resistance to acquire the dye and, therefore, the estequiometry DNA: dye is losing. DNA loss by extrusion during encystment is lastly recovered before the first postexcystment division, restoring the vegetative Ma DNA average content. Using a cytophotometrical method, Chessa and Delmonte Corrado (1994) [9] have reported Ma DNA synthesis during encystment of Colpoda inf/ata. It disagrees with results obtained using inhibitors of DNA synthesis, which do not block the ciliate encystment process [29, 61]. C. inf/ata encystment is not blocked by aphidicolin (a specific inhibitor of a eukaryotic DNA polymerase) and BrdU is not incorporated into

Ciliate nuclear modifications and encystment Ma DNA during encystment (unpublished data), these experiments indicate the absence of Ma DNA synthesis during this process. On the other hand, DNA synthesis inhibitors like hydroxyurea [29] and 5-fluorodeoxyuridine prevent growth and induce encystment in both ciliates and amoebas [74].

Macronuclear DNA Modifications At present, Ma DNA methylation pattern changes are the only type of Ma DNA modification that has been detected during ciliate encystment [54]. DNA dernethylation during eukaryotic cell differentiation has been reported [59], and many studies have established a correlation between undermethylation and unimpeded gene expression. Restriction patterns of vegetative cells and resting cysts of Colpoda inf/ata have shown differences after digestion with HhaI and MspI enzymes, indicating that resting cyst Ma DNA is dernethylated in those sequences with regard to the vegetative stage [54]. Likewise, 5-azacytidine experiments (a potent de methylating agent) corroborate that a possible Ma DNA demethylation takes place during encystment of this ciliate [54]. This experimental evidence involving DNA demethylation during encystment also supports the idea that some specific encystment genes are newly expressed to elaborate the resting stage. The authors [54] believe that activation of encystment specific gene promoters could be possibly achieved by specific Ma DNA demethylation, Furthermore, in Colpoda inf/ata (unpublished data), a methylation in the 18S rDNA has been detected during encystment. A selective methylation on rDNA has also been reported in plants [19], Physarum polycephalum [23], Schizophyllum commune [5] and Tetrahymena thermophila [4], during different development stages. These specific methylations can induce the formation of some unusual DNA structures such as cruciform, curved DNA, intramolecular triplex and left-banded Z DNA [75], it might be another explanation for the results obtained by pulsed field gel electrophoresis with regard to the location of the rDNA subchromosomal band of Colpoda inf/ata resting cysts [55].

Concluding Comments and Future Outlook During ciliate encystment a lot of important macronuclear modifications take place. All these changes are realized to obtain a transcriptionally inactive cryptobiotic nuclear system and to preserve the genetic material from unvafourable environmental conditions. Therefore, the ciliate resting cyst Ma could be an excel-

101

lent microbial eukaryotic model to study different aspects of the gene-silencing mechanism, one of the main characteristics of any microbial cryptobiotic form. However, this aspect of the ciliate encystment is still poorly known and a more extensive analysis at both molecular and structural levels is absolutely necessary. There are still several unresolved questions related with his topic, e.g.: How does the macronuclear fusion mechanism in ciliates with two or more Ma masses occur? What is the role of microtubules in macronuclear fusion and how are they regulated? Are crystal-like chromatin structures a general characteristic in cystic Ma of ciliates? How are they formed? Are specific nuclear proteins involved in Ma chromatin condensation during encystment? What determines the Ma DNA quantity to be removed by extrusion? What is the significance of this DNA loss during encystment? Are there any other DNA modifications, independently of DNA methylation pattern changes, involved in ciliate encystment? What Ma genes are involved in the regulation of the encystment gene expression? These and other unresolved questions should be considered in the future by ciliatologists studying the ciliate encystment nuclear system.

Acknowledgements: This work was supported by grants from Direccion General de Investigacion Cientifica y Tecnica (DGICYT). Projects: PB93-0076 and PB96-0611 to J.c.G., and a predoctoral fellowship from Universidad Complutense (UCM) to S.c.

References

2

3 4

5

6 7

Adl S. M. and Berger J. D. (1997): Timing of life cycle morphogenesis in synchronous samples of Sterkiella histriomuscorum L. The vegetative cell cycle. Europ. J. Protisto!' 33, 99-109. Baroin-Touranchenu A., Tie Y., Lorna 0., and Perasso R. (1996): Informational molecules in encysted Oxytricha. J. Euk. Microbio!. 44, Groupement des Protistologues de Langue Francais 46A. Beers C. D. (1946): History of the nuclei of Tillina magna during division and encystment. J. Morphol78, 181-200. Blackburn E. H., Pan wc, and Johnson C. C. (1983): Methylation of ribosomal RNA genes in the macronucleus of Tetrahymena thermophila. Nucleic Acids Res. 11,5131-5145. Buckner B., Novotny, C. P., and Ullrich R. C. (1988): Developmental regulation of the methylation of the ribosomal DNA in the basidiomycete fungus Schizophyllum commune. Curro Genet. 14, 105-111. Burt R. L., Kidder G. W., and Claff C. L. (1941): Nuclear reorganization in the family Colpodidae. J. Morpho!. 69, 537-561. Calkins G. N. (1930): Uroleptus halseyi Calkins. II. The origin and fate of the macronuclear chromatin. Arch. Protistenkd. 69,151-174.

102

J. C. Gutierrez, A. Martin-Gonzalez and S. Callejas

8 Callejas S. and Gutierrez]. C (1997): Obtencion de sondas de genes de enquistamiento mediante RT-PCR en Oxytricha nova. XVI Congreso S.E.M. Barcelona (Spain). 9 Chessa M. G. and Corrado M. U. D. (1994): Encystment and excystment in Colpoda inflata: Macronuclear DNA and total protein content quantitative variations. Arch. Protistenkd. 144,207-211. 10 Chessa M. G., Corrado M. U. D., and Ramoino P. (1983): Studio de lla fine struttura del macronucleo di Colpoda cucullus. II variare del sistema nucleolare nel corso del ciclo di sviluppo. Estratto dagli Atti dell' Accademia Ligure di Scienze e Lettere. 40, 3-15. 11 Cleffmann G. (1980): Chromatin elimination and the genetic organization of the macronucleus in Tetrahymena thermophila. Chromosoma 78, 313-325. 12 Corliss J. O. (1965): L'autogamie et la senescence du Cilie Hymenostome Tetrahymena rostrata (Kahl), Annee Biologique. 4, 49-69. 13 Corliss]. O. and Esser S. C (1974): Comments on the role of the cyst in the life cycle and survival of free-living protozoa. Trans. Am. Microsc. Soc. 93, 578-593. 14 Dallai R., Luporini P., and Miceli C (1985): Macronuclear inclusions in the ciliated protozoon Euplotes. Trans. Am. Microsc. Soc. 104,64-69. 15 Dallai R, Miceli C, and Luporini P. (1987): Euplotes rariseta Curds et al. (Ciliophora Hypotrichida) from the somalian coast: Description and preliminary observations on cyst induction and ultrastructure. Italian J. Zool. 21, 263-280. 16 Diaz S., Martin-Gonzalez A., and Gutierrez]. C (1995): Estudio morfogenetico de la division y enquistamiento del ciliado colpodido Cyrtolophosis elongata. XV Congreso SEM. Madrid (Spain). 17 Dodd E. E. (1962): Cytochemical investigation of the nuclear changes in the cyst cycle of the ciliate Telotrochidiurn henneguyi. J. Protozool. 9, 93-97. 18 Faure-Fremiet E. (1953): L'hypothese de la senescence et les cycles de reorganisation nucleaire chez les Cilies, Rev. Suisse Zool. 60, 426-438. 19 Finnegan E. J., Brettell R. I. S., and Dennis E. S. (1993): The role of DNA methylation in the regulation of plant gen expression. In: DNA methylation: Molecular Biology and Biological Significance. J. P. Jost and H. P. Saluz eds., 218-261. Birkhauser Verlag Basel. Switzerland. 20 Foissner I. and Foissner W. (1987): The fine structure of the resting cysts of Kahliella simplex (Ciliata, Hypotrichida). Zool. Anz. 218, 65-74. 21 Fenkel M. A. (1980): Fine structure of the macronucleus of the active and encysted (dividing) forms of ciliate Colpoda steinii. Protistologica 16,339-351. 22 Frenkel M. A. (1992): Fine structure of the macronucleus in the resting cysts of the ciliate Tillina magna. Arch. Protistenkd. 141,27-40. 23 Fronk ]. and Magiera R. (1994): DNA methylation during differentiation of a lower eukaryote, Physarum polycephalum.J. Biochem. 304, 101-104. 24 Garnjobst L. (1928): Induced encystment and excystment in Euplotes taylori sp. nov. Physiological Zool. 1, 561-575. 25 Grimes G. W. (1973): Differentiation during encystment and excystment in Oxytricha fallax. J. Protozool. 20, 92-104. 26 Gutierrez J. C (1985): Quantitative cytochemical study of chromatin and histones on isolated macronuclear masses from the resting cysts of Gastrostyla steinii. Microbios. 43,43-51.

27 Gutierrez J. C and Perez-Silva J. (1983): Ultrastructural aspects of the precystic and cystic cytoplasm of the Hypotrichous Ciliate, Laurentiella acuminata. Acta Protozool. 22, 203-210. 28 Gutierrez]. C, Martin-Gonzalez A., and Matsusaka T. (1990): Towards a generalized model of encystment (cryptobiosis) in ciliates: A review and a hypothesis. BioSystems 24,17-24. 29 Gutierrez J. C, Torres A., and Perez-Silva J. (1981): Excystement cortical morphogenesis and nuclear processes during encystment and excystment in Laurentiella acuminata (Hypotrichida, Oxytrichidae). Acta Protozool. 20, 145-152. 30 Gutierrez]. C, Torres A., and Perez-Silva J. (1983): Fine structure of the cyst wall of Laurentiella acuminata (Hypotrichida: Oxytrichidae). Trans. Am. Micros. Soc. 102, 55-59. 31 Jareiio M. A. (1985): Etude ultrastructurale de l'enkystement et du dekystement chez Onychodromus acuminatus (Ciliate, hypotrichida). Protistologica 21, 313-321. 32 Kawakami H. and Yagiu R. (1963): The electron microscopical study of the change of fine structure in the ciliate, Colpoda cucullus, during its life cycle. III. From the stage of completion of the first layer of resting cyst membrane to the completion of the resting cyst. Zool. Mag. 72, 224-229. 33 Kay M. W. (1945): Studies on Oxytricha bifaria stokes. II. Cystic reorganization. Trans. Am. Microsc. Soc. 64, 267-282. 34 Keilin D. (1959): The problem of anabiosis or latent life: History and current concept. Proc. Roy. Soc. London B150, 149-191. 35 Kidder G. W. (1933): On the genus Ancistruma Strand (Ancistrum Maupas). I. The structure and division of A. mytitli Quenn. and A. Isseli Kahl. BioI. Bull. 64,1-20. 36 Kidder G. W. and Claff C L. (1938): Cytological investigations of Colpoda cucullus. BioI. Bull. 74, 178-197. 37 Kink]. (1973): The organization of fibrillar structures in the trophic and encysted Dileptus visscheri (Ciliata, Rhabdophorina). Acta Protozool. 12, 173-191. 38 Leforestier A. and Livolant F. (1993): Supramolecular ordering of DNA in the cholesteric liquid crystalline phase: An ultrastructural study. Biophysical J. 65, 56-72. 39 Leforestier A. and Livolant F. (1994): DNA liquid crystalline blue phases electron microscopy evidence and biological implications. Liquid Crystals 17,651-658. 40 Lieder H. J. and Ruthmann A. (1976): Cytological and cytophotometric studies of the macronucleus of a hypotrichous ciliate of the genus Oxytricha. Arch. Protistenkd. 118,268-284. 41 Lipps H. J. (1980): In vitro aggregation of the gene-sized DNA molecules of the ciliate Stylonychia mytilus. Proc. Nat!. Acad. Sci. USA 77, 4104-4107. 42 Manwell R. D. (1928): Conjugation, division, and encystment in Pleurotricha lanceolata. Biol, Bull. 54, 417-463. 43 Martin-Gonzalez A., Benitez L., and Gutierrez]. C (1991): Cortical and nuclear events during cell division an resting cyst formation in Colpoda inflata. J. Protozool. 38,336-344. 44 Martin-Gonzalez A., Benitez L., and Gutierrez J. C (1992): Ultrastructural analysis of resting cyst and encystment in Colpoda inflata 2 Encystment process and a review of ciliate resting cyst classification. Cytobios 72, 93-106.

Ciliate nuclear modifications and encystment

45 Martin-Gonzalez A., Palacios G., and Gutierrez J. C. (1994): Cyst wall precursors of Colpoda inf/ata: A comparative ultrastructural study and a review of ciliate cyst wall precursors. Cytobios. 77, 217-223. 46 Martin-Gonzalez A., Palacios G., and Gutierrez J. C. (1995): Estudio de la actividad fosfatasa in situ durante el enquistamiento del ciliado Colpoda inf/ata. XV Congreso S.E.M. Madrid. 47 Matsusaka T. (1976): An ultrastructural study of encystment in the hypotrichous ciliate Pleurotricha sp. Kumamoto J. Sci. BioI. 13, 13-26. 48 Matsusaka T (1979): Effect of cycloheximide on the encystment and ultrastructure of the ciliate Histriculus. J. Protozoo I. 26, 619-625. 49 Matsusaka T. and Kimura S. (1981): Changes in macronuclear ultrastructures during encystment in a ciliate, Histriculus muscorum. Kumamoto J. Science BioI. 15, 49-58. 50 McArdle E. w., Bergquist B. L., and Ehret C. E (1980): Structural changes in Tetrahymena rostrata during induced encystment. J. Protozool. 27, 388-397. 51 Melese T. and Xue Z. (1995): The nucleolus: an organelle formed by the act of building a ribosome. Curro Opin. Cell. BioI. 7, 319-324. 52 Morat G., Chessa G., and Crippa-Franchesi T. (1981): Etude de la regulation des teneours en ADN nucleaire au cours de la reproduction vegetative et L'enkystement d'attente chez Ie cilie Colpoda cucullus. Europ. J. Protistol. 17, 313-329. 53 Padnos M. (1962): Cytology of cold induced transformation of ectogenic reproductive cysts to resting cysts in Colpoda maupasi. J. Protozool. 9,13-20. 54 Palacios G., Martin-Gonzalez A., and Gutierrez J. C. (1994): Macronuclear DNA demethylation is involved in the encystment process of the ciliate Colpoda inf/ata. Cell BioI. Int. 18,223-228. 55 Palacios G., Martin-Gonzalez A., and Gutierrez J. C. (1995): Macronuclear chromatin study in vegetative cells and resting cyst of Colpoda inf/ata: Chromatin spreading and standard transmission electron microscopy. Second European Congress of Protistology and Eighth European Conference on Ciliate Biology. Clermont-Ferrand (France). 56 Raabe H. (1947): L'appareil nucleaire d'Urostyla grandis EHRBG.II. Appareil macronuclaire. Ann. Univ, M. Curie-Sklodowska Cl, 133-170. 57 Raikov 1. B. (1982): The Protozoan Nucleus, Morphology and Evolution. Springer-Verlag, New York. 58 Raikov 1. B. (1995): Structure and genetic organization of the polyploid macronucleus of ciliates: A comparative review. Acta Protozool. 34, 151-171. 59 Razin A. and Cedar H. (1991): DNA methylation and gene expression. Microbiol. Reviews 55, 451-458. 60 Rios R. M., Torres A., Calvo P, and Fedriani C. (1985): The cyst of Urostyla grandis (Hypotrichida: Urostylidae): Ultrastructure and evolutionary implications. Protistologica 21, 481-485. 61 Ruthmann A. and Kuck A. (1985): Formation of the cyst wall of the ciliate Colpoda steinii. J. Protozool. 32, 677-682.

103

62 Sergejeva G. 1. and Bobyleva N. N. (1988): The formation of crystal-like structures in somatic nucleus chromatin of Bursaria (Ciliophora, Protozoa) upon their preparation to a longterm resting state. Tsitologia 30, 1291-1299. (In Russian with English summary). 63 Sergejeva G. 1., Bobyleva N. N., and Ibrahimov R. K. (1987): Electron microscopic study of somatic nucleus chromatin at different stages of encystment in the ciliate Bursaria truncatella. Tsitologia 29, 5-10. (In Russian with English summary). 64 Tikhonenko A. S., Bespalova 1. A., Martinkina L. P, Popenko V. 1., and Sergejeva G. 1. (1984): Structural organization of macro nuclear chromatin of the ciliate Bursaria truncatella in resting cysts and at excysting. Europ. J. Cell BioI. 33, 37-42. 65 Tittler J. A. (1935): Division, encystment and endomixis in Urostyla grandis, with an account of an arnicronucleatc race. La Cellule 44,189-218. 66 Verni E, Rosati G., and Ricci N. (1984): The cyst of Oxytricha bifaria (Ciliata Hypotrichida). II. The ultrastructure. Protistologica 20,87-95. 67 Wada R. K. and Spear B. B. (1980): Nucleosomal organization of macronuclear chromatin in Oxytricha fallax. Cell Differentation 9, 261-268. 68 Walker G. K. and Goode D. (1976): The role of microtubules in macronuclear division in the hypotrich ciliates Gastrostyla steinii and Stylonychia mytilus. Cytobiologie 14,18-37. 69 Walker G. K. and Hofmman J. T. (1985): An ultrastructural examination of cyst structure in the hypotrich ciliate Gonostomum species. Cytobios 44,153-161. 70 Walker G. K. and Maugel T. K. (1980): Encystment and excystment in hypotrich ciliates. II. Diophrys scutum and remarks on comparative features. Protistologica 16, 525-531. 71 Walker G. K., Edwards C. A., and Suchard S. J. (1989): Encystment in the peritrich ciliate Telotrochidium henneguyi. Cytobios 59, 7-18. 72 Walker G. K., Maugel T. K., and Goode D. (1975): Some ultrastructural observations on encystment in Styponychia mytilus (Ciliophora: Hypotrichida). Trans. Am. Microsc. Soc. 94, 147-154. 73 Walker G. K., Maugel T. K., and Goode D. (1980): Encystment and excystment in hypotrich ciliates. 1. Gastrostyla stein ii, Protistologica 16, 511-524. 74 Weisman R. A. (1976): Differentiation in Acanthamoeba castellanii. Ann. Rev. Microbiol. 30, 189-219. 75 Zacharias W. (1993): Methylation of cytosine influences the DNA structure. In: DNA Methylation: Molecular Biology and Biological Significance. J. P.Jost and H. P. Saluz eds. pp 27-38. Birkhauser Verlag Basel. Switzerland.

Address for correspondence: Dr. Juan Carlos Gutierrez, Departamento de Microbiologia-III, Facultad de Biologia, Universidad Complutense (UCM), 28040 Madrid, Spain; Fax # 3944964; E-mail: [email protected];[email protected]