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

Minireview

European Journal of

PROTISTOLOGY

Nuclear Changes, Macronuclear ChromatinReorganization and DNA Modifications duringCiliate 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 aspectsof ciliate encystment; the nuclear and chromatin changesinvolved 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: thegene-silencing and the genome preservation. The nuclearchanges include: macronuclear fusion and chromatin con­densation, formation of chromatin crystal-like structuresin some ciliates, nucleolar fusion and rONA inactivation,macronuclear DNA loss and specific DNA modifications.

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

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 truecryptobiosis phenomenon according to Keilin's defini­tion [34]. Excystment is the process of emergence fromthe cryptobiotic stage (resting cyst). Together theseprocesses 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 volumeloss 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 derivedfrom different precursors [30, 45, 48, 70], organellesclustering as a consequence of cytoplasmic dehydration[27, 47, 69], a high autophagic activity [46] and drastic

© 1998 by GustavFischer Verlag

nuclear changes [28]. Here we will consider only oneaspect of ciliate encystment, the nuclear changes. Nu­clear dualism is a general feature of ciliates and changesin both kinds of nuclei have been reported during cili­ate encystment. These changes include: macronuclear(Ma) chromatin condensation, Ma-DNA loss, changesin the Ma-DNA methylation pattern, nucleolarchanges and an extensive rearrangement of the Machromatin. All these modifications probably serve tointerfere with gene expression, and are indicative oftranscriptional inactivity, the main characteristic of anycryptobiotic form.

Macronuclear Fusion and/or ChromatinCondensation

In general, during encystment, macronuclei in cili­ates with several macronuclei (Ma) in the vegetativestage fuse to form only one cystic macronuclear (Ma)mass. This process has been reported mainly in sti­chotrichs and hypotrichs ciliates, such as Gonostomumsp. [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 thisrule has been observed in the stichotrich Pleurotrichasp. [47] and Urostyla grandis which have many smallMa in vegetative cells and resting cysts [60]. In the lastspecies, several Ma degenerate during encystment, sothe resting cyst has a lower number of Ma than the veg­etative cell [65]. Macronuclear fusion causes a drasticvolume reduction and chromatin condensation. Inthose 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 issimilar to the condensation that takes place in prepara­tion for division during the Gz period in ciliates withmultiple Ma, or during physiological reorganization orposttraumatic regeneration of ciliates [57]. All these in­stances of Ma fusion are correlated with some corticalchanges, e.g. buccal reorganization, cortical divisionand/or total or partial kinetosome reabsorbtion duringencystment.

Some authors suggest that Ma chromatin is redis­tributed and that the configuration of chromatin ele­ments changes when the Ma condenses [57]. It couldfavour the homogeneous repartition of genetic materialbetween both daughter cells during division. Besides aMa reorganization, Ma fusion during encystment couldhave another meaning: to maintain constant the nucleo­cytoplasmic ratio in the cell. A high correlationbetween the cytoplasmic and Ma volume loss has beenfound in some ciliates [26, 44]. If this correlation isobserved 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, Urostylagrandis, an exception among stichotrich ciliates with­out Ma fusion during encystment [60], shows Ma fu­sion of its over 100 Ma during cell division [56]. In thiscase, the nucleo-cytoplasmic ratio could be maintainedby degeneration of several Ma during encystment [65].

How does Ma fusion occur? At present, we knowvery little about the elements involved in the Ma fusionand how they work. In both division and encystment,microtubules are present during Ma fusion. High num­bers of microtubules seem to be involved in severalmacronuclear changes during stichotrich cortical mor­phogenesis, including: Ma fusion by enlargement of in­terconnecting isthmuses, mixing of the Ma chromatinduring condensation and elongation, and Ma division[68]. During encystment a high microtubular density inboth 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 severalhypotheses for the function of microtubules during en­cystment [73]: Microtubules may function passively byproviding a rigid karyoskeletal guide for the fusing Maor they may actively widen the isthmus joining the Ma.Likewise, the microtubules, via their insertions into thenuclear envelope and chromatin, could provide the mo­tive force responsible for the chromatin condensation[73]. This last hypothesis has been used to explain thepresence 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 inthe encystment Ma fusion and/or condensation. Thepresence of Ma microtubules has also been reportedduring excystment [31, 73], they may have an impor­tant function during the excystment Ma amitotic divi­sion that occurs in some ciliates. Macronuclei are themain nuclear elements undergoing condensation dur­ing encystment because micronuclear (Mi) chromatincondensation has been only reported in few ciliates [37,72]. Macronuclear chromatin condensation is accompa­nied by drastic changes in chromatin organization andultrastructure, e.g., in stichotrich ciliates, Oxytrichafal/ax [25], Histriculus muscorum [49], Laurenttellaacuminata [27], Gastrostyla steinii [73], Stylonychiamytilus [72], Pleurotricha sp. [47] and O. hifaria [66],large spheroidical bodies are usual Ma chromatinicstructures in the resting cysts. In general, an increase insize of Ma chromatin bodies is reported during encyst­ment [15, 20, 32, 55], which is due to the fusion ofsmaller chromatin bodies. This compactation is special­ly high in some colpodid resting cysts, Bursaria trun­catella [63], B. ovata [62] and Colpoda inf/ata [55]. Inthese species, crystal-like hexagonal chromatin struc­tures (liquid crystal type) have been observed in the Machromatin of resting cysts, after the application ofchromatin spreading procedures. Probably, the highlevel of dehydration that the cell undergoes during en­cystment is an important factor in the formation ofthese polygonal structures. Spontaneous DNA order­ing into liquid crystalline phases at high concentrationhas been hypothesized as an important mechanism inchromatin packaging [39]. These liquid crystaline phas­es, which depend on the polymer concentration, can befound in vitro and in vivo, e.g., hexagonal packing ofDNA molecules (columnar hexagonal phase) wasfound in bacteriophages and sperm nuclei [38]. The for­mation of this liquid crystalline chromatin organiza­tion, reported in several organisms, is presumably dueto both, condensation and macromolecular dehydra­tion. These Ma chromatin crystalline hexagonal struc­tures, only found until now in colpodid ciliates, may bea resting cyst specific form of chromatin packing. Be­sides, crystal-like or paracrystaline bodies are com­monly found in both cytoplasm and macronucleus ofencysted ciliates [14, 27], which are formed by the celldehydration.

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 onestudy on this topic using Gastrostyla steinii [26], inwhich a micro spectrophotometric analysis reveals thatthe cystic Ma had about 1.23-fold more histones than a

single vegetative Ma mass and that these cystic Ma his­tones were about 1.59-fold more arginine-rich than thevegetative macronuclear ones. This may be suggestingthat the high Ma DNA condensation in resting cystscould be due to the presence of arginine-rich proteins(such as protamines), as in metazoan cells where substi­tution of protamines for histones in spermatogenesis iscorrelated with an extremely dense chromatin packingand loss of RNA synthetic capacity. However, a studyof nucleosomes spacing in the chromatin of Oxytrichafallax [67] has shown that the vegetative spacing with arepeat length of 198 bp is maintained in the resting cyst.The Ma chromatin condensation during encystmentmay be involved in the transcriptional inactivation ofthe resting stage.

Another example of molecular DNA aggregation isprobably 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 vegetativecells) is almost completely restrained to one big elec­trophoretic band (>20 Kb size) in the resting cyst MaDNA. This molecular aggregation could be similar tothe in vitro aggregation of the DNA molecules report­ed in Stylonychia mytilus [41], obtained by incubatingthe Ma DNA under increasingly stronger ionic condi­tions, because the electrophoretic patterns look verysimilar.

Other nuclear changes involved in ciliate encystmentinclude: nuclear envelope changes, modification of thepattern of nuclear pores, micronuclear degradation andautogamy. Changes to both, nuclear envelope and/ornucleopores have been shortly studied. During encyst­ment in Euplotes rariseta [15] there are drastic changesin nuclear pore number and distribution pattern. Thehexagonal pattern of vegetative nucleopores changes toa lineal distribution in the resting cyst and their numberis considerably reduced. During excystment the vegeta­tive number of nucleopores is recovered, as shown inOnychodromus acuminatus [31]. This reduction in thenumber 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 inseveral ciliates [27, 37, 72, 73]. This could representprotection against the high autophagosomic activityduring encystment. In fact, a Mi number reduction, dueto Mi degradation during encystment, has been report­ed in some ciliates, e.g. Laurentiella acuminata [29],Gastrostyla steinii [73] and Oxytricha fallax [25]. Thevegetative average Mi number is restored by mitosisduring excystment [25,29], and a Mi DNA synthesisprevious to mitosis has been observed [29]. In thesespecies the excystment Mi mitosis is coincident withthe amitotic Ma division that restores the average num­ber of vegetative Ma masses.

Ciliate nuclear modifications and encystment 99

Sexual processes (autogamy) have been only report­ed during encystment in Tetrahymena rostrata [12,26].The encystment of this ciliate can be divided in twophases, a first pre-autogamic phase in which the cellforms the cyst wall from mucocyst secretion [50] and asecond phase in which the autogamy takes place. Froma 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 resistancemechanism against unfavourable environmental condi­tions (resting cyst).

Nucleoli and Encystment

In general, nucleolar structure changes take place inthe cell as response to many external factors and as aconsequence 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 nucleolioccur during encystment, which indicates importantchanges in the rRNA metabolism. Three main types ofnucleolar modifications can be distinguished duringciliate encystment: structural modifications with nucle­olar fusion (Colpoda cucullus [32], C. inf/ata f55], Tilli­na magna [22] and Dileptus visscheri [37]), structuralmodifications without fusion (Stylonychia mytilus [72],Gastrostyla steinii [73], Oxytricha fallax [25] andHistriculus 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] anynucleolar alteration has been reported.

Nucleolar morphology undergoes drastic changesdepending on physiological state of the cell [51]. Forexample, in Paramecium [57] and in Tetrahymena [57,58] the multiple nucleoli fuse under starvation or sta­tionary phase conditions. Similar changes occurs inTetrahymena under the action of RNA synthesis in­hibitor Actinomycin D, cadmium ions or ultravioletirradiation [58]. We may find a short parallelism amongthese 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 thegranular nucleolar component observed in the precys­tic cells of some ciliates may be explained by the neces­sity for rRNA synthesis for the cyst wall molecularbiosynthesis. In late precystic phases the biosyntheticactivity decreases and so the rRNA requirement wouldbe lower, which may explain the drastic ultrastructuralchanges that nucleoli undergo during encystment, suchas disappearance or condensation. Probably, nucleolar

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

changes are connected with the Ma chromatin changesresulting 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 thebiosynthetic ribosomal mechanism may be related withthe rDNA subchromosomal location in the resting cystpulsed field gel electrophoretic pattern [55], and thismolecular observation might confirm the nucleolarinactivation in resting cysts by condensation orpacking.

Macronuclear Extrusion Bodies

Macronuclear extrusion occurs in both, division andencystment. It may take place during the Ma division ofTetrahymena, Colpidium [57], Urocentrum turbo, An­cistruma isseli, Allosphaerium convexa and others [6],or after Ma division, such as in division cysts of manycolpodida [6, 21, 36, 43]. Like in cell division, a Machromatin extrusion occurs during encystment ofmany colpodid ciliates [3, 6, 22, 36, 43, 53], but not inothers as Colpoda aspera [6] and Cyrtolophosis elongata[16]. In general, Ma extrusion involves the formation ofonly one extrusion body, but in some cases more thanone extrusion body is formed during encystment, e.g.Colpoda maupasi undergoes double extrusion duringthe cold induced encystment [53], and in Tillina magna,the extrusion occurs repeatedly during the encystmentprocess [22]. Chromatin extrusion is not very commonin 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 oflost Ma DNA is very variable among cells of a hetero­geneous encysting population. A flow cytometry DNAstudy in C. inf/ata (unpublished data) has shown thatabout 47% of the Ma DNA is lost during encystment.This amount represents the average Ma DNA amountof an extrusion body.

What type of Ma DNA is lost? Is Ma extrusion anunspecific or specific process? The few data availableon these questions indicate that the extrusion processdoes not eliminate exclusively rDNA, as it was pro­posed by Fenkel (1980) [21], rather it is an unspecificprocess 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 autoradiographicdata have shown that the DNA extrusion body doesnot differ from the bulk Ma DNA [57], and, conse-

quently, it can not be considered a specific DNA, suchasrDNA.

At present, we do not know how this process is dis­charged and how it is regulated. Several hypothesesconcerning to explain the physiological significance ofthis DNA loss during both division and encystmenthave been presented. In 1930, Calkins [7] explained it asa "purification" process and Kidder (1933) [35] namedit "cleaning" macronuclear process. Kidder and Claff(1938) [36] considered that the DNA reorganization in­volved in the extrusion body formation replaced theabsence of conjugation in these ciliates. Faure-Fremiet(1953) [18] supposes that this extrusion process is a reg­ulation mechanism of macronuclear polyploidy, byeliminating the "extra" genomes or maintaining thechromosomic macronuclear equilibrium by extrusionof "extra" chromosomes. In Tetrahymena thermophila[11] the DNA content of the division extrusion bodiesis not a multiple of the Mi DNA content, so the chro­matin extrusion can not be considered the eliminationof whole genomes from Ma. According to another hy­pothesis, chromatin extrusion is a means to regulate thenucleocytoplasmic ratio [57]. After division and en­cystment, the cell volume is reduced and as the nucleo­cytoplasmic ratio must be maintained, the nuclearvolume must be also reduced, and it can be obtained bychromatin condensation and/or chromatin extrusion.We think that perhaps this last assumption is the mostappropriate, taking into account the strong correlationbetween 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 formationcould be also occur during encystment. In fact, inOxytricha sp. [40] a reduction of the total Ma DNA toabout the half of the average content has been reported.However, we must be circumspect when we explaindata 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 acquirethe dye and, therefore, the estequiometry DNA: dye islosing.

DNA loss by extrusion during encystment is lastlyrecovered 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 DNAsynthesis, which do not block the ciliate encystmentprocess [29, 61]. C. inf/ata encystment is not blockedby aphidicolin (a specific inhibitor of a eukaryoticDNA polymerase) and BrdU is not incorporated into

Ma DNA during encystment (unpublished data), theseexperiments indicate the absence of Ma DNA synthesisduring this process. On the other hand, DNA synthesisinhibitors like hydroxyurea [29] and 5-fluorodeoxyuri­dine prevent growth and induce encystment in bothciliates and amoebas [74].

Macronuclear DNA Modifications

At present, Ma DNA methylation pattern changesare the only type of Ma DNA modification that hasbeen detected during ciliate encystment [54]. DNAdernethylation during eukaryotic cell differentiationhas been reported [59], and many studies have estab­lished a correlation between undermethylation andunimpeded gene expression. Restriction patterns ofvegetative cells and resting cysts of Colpoda inf/atahave shown differences after digestion with HhaI andMspI enzymes, indicating that resting cyst Ma DNA isdernethylated in those sequences with regard to thevegetative stage [54]. Likewise, 5-azacytidine experi­ments (a potent demethylating agent) corroborate thata possible Ma DNA demethylation takes place duringencystment 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 restingstage. The authors [54] believe that activation of encyst­ment specific gene promoters could be possiblyachieved 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 hasalso been reported in plants [19], Physarum poly­cephalum [23], Schizophyllum commune [5] andTetrahymena thermophila [4], during different devel­opment stages. These specific methylations can inducethe formation of some unusual DNA structures such ascruciform, curved DNA, intramolecular triplex andleft-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 rDNAsubchromosomal 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 arerealized 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-

Ciliate nuclearmodifications and encystment 101

lent microbial eukaryotic model to study different as­pects of the gene-silencing mechanism, one of the maincharacteristics of any microbial cryptobiotic form.However, this aspect of the ciliate encystment is stillpoorly known and a more extensive analysis at bothmolecular and structural levels is absolutely necessary.

There are still several unresolved questions relatedwith his topic, e.g.: How does the macronuclear fusionmechanism in ciliates with two or more Ma massesoccur? What is the role of microtubules in macronucle­ar fusion and how are they regulated? Are crystal-likechromatin structures a general characteristic in cysticMa of ciliates? How are they formed? Are specific nu­clear proteins involved in Ma chromatin condensationduring encystment? What determines the Ma DNAquantity to be removed by extrusion? What is the sig­nificance of this DNA loss during encystment? Arethere any other DNA modifications, independently ofDNA methylation pattern changes, involved in ciliateencystment? What Ma genes are involved in the regula­tion of the encystment gene expression? These andother unresolved questions should be considered in thefuture by ciliatologists studying the ciliate encystmentnuclear system.

Acknowledgements: This work was supported by grantsfrom 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

Adl S. M. and Berger J. D. (1997): Timing of life cyclemorphogenesis in synchronous samples of Sterkiellahistriomuscorum L. The vegetative cell cycle. Europ. J.Protisto!' 33, 99-109.

2 Baroin-Touranchenu A., Tie Y., Lorna 0., and Perasso R.(1996): Informational molecules in encysted Oxytricha. J.Euk. Microbio!. 44, Groupement des Protistologues deLangue Francais 46A.

3 Beers C. D. (1946): History of the nuclei of Tillina magnaduring division and encystment. J. Morphol78, 181-200.

4 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.

5 Buckner B., Novotny, C. P., and Ullrich R. C. (1988): De­velopmental regulation of the methylation of the riboso­mal DNA in the basidiomycete fungus Schizophyllumcommune. Curro Genet. 14, 105-111.

6 Burt R. L., Kidder G. W., and Claff C. L. (1941): Nuclearreorganization in the family Colpodidae. J. Morpho!. 69,537-561.

7 Calkins G. N. (1930): Uroleptus halseyi Calkins. II. Theorigin 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 enOxytricha nova. XVI Congreso S.E.M. Barcelona (Spain).

9 Chessa M. G. and Corrado M. U. D. (1994): Encystmentand excystment in Colpoda inflata: Macronuclear DNAand 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 Colpodacucullus. II variare del sistema nucleolare nel corso delciclo di sviluppo. Estratto dagli Atti dell' AccademiaLigure di Scienze e Lettere. 40, 3-15.

11 Cleffmann G. (1980): Chromatin elimination and the ge­netic organization of the macronucleus in Tetrahymenathermophila. Chromosoma 78, 313-325.

12 Corliss J. O. (1965): L'autogamie et la senescence du CilieHymenostome Tetrahymena rostrata (Kahl), Annee Bio­logique. 4, 49-69.

13 Corliss]. O. and Esser S. C (1974): Comments on the roleof 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): Macronuclearinclusions in the ciliated protozoon Euplotes. Trans. Am.Microsc. Soc. 104,64-69.

15 Dallai R, Miceli C, and Luporini P. (1987): Euplotesrariseta Curds et al. (Ciliophora Hypotrichida) from thesomalian coast: Description and preliminary observationson 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 enquistamientodel 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 etles 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 plantgen expression. In: DNA methylation: Molecular Biologyand 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 ofthe resting cysts of Kahliella simplex (Ciliata, Hypotrichi­da). Zool. Anz. 218, 65-74.

21 Fenkel M. A. (1980): Fine structure of the macronucleusof the active and encysted (dividing) forms of ciliateColpoda steinii. Protistologica 16,339-351.

22 Frenkel M. A. (1992): Fine structure of the macronucleusin the resting cysts of the ciliate Tillina magna. Arch. Pro­tistenkd. 141,27-40.

23 Fronk ]. and Magiera R. (1994): DNA methylation duringdifferentiation of a lower eukaryote, Physarum polyce­phalum.J. Biochem. 304, 101-104.

24 Garnjobst L. (1928): Induced encystment and excystmentin Euplotes taylori sp. nov. Physiological Zool. 1, 561-575.

25 Grimes G. W. (1973): Differentiation during encystment andexcystment in Oxytricha fallax. J. Protozool. 20, 92-104.

26 Gutierrez J. C (1985): Quantitative cytochemical study ofchromatin and histones on isolated macronuclear massesfrom the resting cysts of Gastrostyla steinii. Microbios.43,43-51.

27 Gutierrez J. C and Perez-Silva J. (1983): Ultrastructuralaspects 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 processesduring encystment and excystment in Laurentiella acumi­nata (Hypotrichida, Oxytrichidae). Acta Protozool. 20,145-152.

30 Gutierrez]. C, Torres A., and Perez-Silva J. (1983): Finestructure 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 stageof completion of the first layer of resting cyst membraneto 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. LondonB150, 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 inthe 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 andcytophotometric 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-sizedDNA 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 anresting 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.

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 cystwall precursors. Cytobios. 77, 217-223.

46 Martin-Gonzalez A., Palacios G., and Gutierrez J. C.(1995): Estudio de la actividad fosfatasa in situ durante elenquistamiento del ciliado Colpoda inf/ata. XV CongresoS.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 organelleformed 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 aucours 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 inColpoda maupasi. J. Protozool. 9,13-20.

54 Palacios G., Martin-Gonzalez A., and Gutierrez J. C.(1994): Macronuclear DNA demethylation is involved inthe encystment process of the ciliate Colpoda inf/ata. CellBioI. Int. 18,223-228.

55 Palacios G., Martin-Gonzalez A., and Gutierrez J. C.(1995): Macronuclear chromatin study in vegetative cellsand resting cyst of Colpoda inf/ata: Chromatin spreadingand standard transmission electron microscopy. SecondEuropean Congress of Protistology and Eighth Euro­pean Conference on Ciliate Biology. Clermont-Ferrand(France).

56 Raabe H. (1947): L'appareil nucleaire d'Urostyla grandisEHRBG.II. Appareil macronuclaire. Ann. Univ, M.Curie-Sklodowska Cl, 133-170.

57 Raikov 1.B. (1982): The Protozoan Nucleus, Morphologyand Evolution. Springer-Verlag, New York.

58 Raikov 1. B. (1995): Structure and genetic organization ofthe polyploid macronucleus of ciliates: A comparative re­view. Acta Protozool. 34, 151-171.

59 Razin A. and Cedar H. (1991): DNA methylation andgene 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 cystwall of the ciliate Colpoda steinii. J. Protozool. 32,677-682.

Ciliate nuclear modifications and encystment 103

62 Sergejeva G. 1.and Bobyleva N. N. (1988): The formationof crystal-like structures in somatic nucleus chromatin ofBursaria (Ciliophora, Protozoa) upon their preparationto a longterm resting state. Tsitologia 30, 1291-1299. (InRussian with English summary).

63 Sergejeva G. 1., Bobyleva N. N., and Ibrahimov R. K.(1987): Electron microscopic study of somatic nucleuschromatin at different stages of encystment in the ciliateBursaria truncatella. Tsitologia 29, 5-10. (In Russian withEnglish 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 Bursariatruncatella in resting cysts and at excysting. Europ. J. CellBioI. 33, 37-42.

65 Tittler J. A. (1935): Division, encystment and endomixisin Urostyla grandis, with an account of an arnicronucleatcrace. La Cellule 44,189-218.

66 Verni E, Rosati G., and Ricci N. (1984): The cyst ofOxytricha 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 ciliatesGastrostyla steinii and Stylonychia mytilus. Cytobiologie14,18-37.

69 Walker G. K. and Hofmman J. T. (1985): An ultrastruc­tural examination of cyst structure in the hypotrichciliate Gonostomum species. Cytobios 44,153-161.

70 Walker G. K. and Maugel T. K. (1980): Encystment andexcystment in hypotrich ciliates. II. Diophrys scutum andremarks 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): Someultrastructural 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 steinii, Protistologica 16, 511-524.

74 Weisman R. A. (1976): Differentiation in Acanthamoebacastellanii. Ann. Rev. Microbiol. 30, 189-219.

75 Zacharias W. (1993): Methylation of cytosine influencesthe DNA structure. In: DNA Methylation: Molecular Bi­ology and Biological Significance. J. P.Jost and H. P.Saluzeds. 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: juancar@eucmax.sim.ucm.es;jgf00004@teleline.es