Stomatogenesis during sexual and asexual reproduction in an amicronucleate strain of Paramecium caudatum.

Amicronucleate cells of Paramecium caudatum, whose micronuclei have been artifically removed by micropipetting, are characterized by the appearance of a deciliated area at the posterior part of the buccal opening. These cells form food vacuoles at a slightly lower rate than micronucleate cells. Their mean interfission time is longer than that in micronucleates. The exconjugants of amicronucleate cells can not form food vacuoles and eventually die witout fission, though conjugation proceeds normally in them as well as in their micronucleate mate. The oral apparatus of amicronucleate exconjugants seems to be shallower than that of micronucleates. The membranellar cilia, therefore, can be seen through the buccal overture by scanning electron microscope. The results obtained from the cross of micronucleate and amicronucleate strains and from the induction of autogamy in amicronucleate strains suggest that the micronucleus has a primary role in developing the normal oral apparatus after nuclear reorganization.     

Evidence for Micronuclear Function During Vegetative Growth and Reproduction of the Ciliate, Tetrahymena pyrijormis

An amicronucleate clone of Tetrahymena pyrijormis has been found among the asexual progeny of irradiated cells of strain EU 6000 (variety 6, mating type I). Log-phase cells of this clone, designated EU 6525, have a mean generation time (6.0 hr) longer than that of the micronucleate strain, EU 6000 (2.9 hr). Further irradiation studies of strain EU 6000 indicate that the recovery of viable amicronucleate populations is rare although many amicronucleate cells are found among surviving progeny.¹ Attempts to introduce micronuclei into amicronucleate cells of strain EU 6525 by conjugation have been made. Micronucleate lines are obtained from amicronu create pair members only in low frequency. These results, considered together with those of other workers, suggest that some change in the state of the cell, additional to the physical loss (or gain) of the micronucleus, must occur before viable amicronucleate clones can be obtained from micronucleate cells, or before amicronucleate cells can produce viable micronucleate lineages. An alteration in mean generation time may be a reflection of this change, or it may simply be a direct consequence of micronuclear removal. The results further imply that the ciliate micronucleus unquestionably contributes information to the cell during asexual growth and reproduction.     

Inactivation of a macronuclear intra-S-phase checkpoint in Tetrahymena thermophila with caffeine affects the integrity of the micronuclear genome

Aphidicolin (APH), an inhibitor of DNA polymerase α, arrested cell divisions in Tetrahymena thermophila. Surprisingly, low concentrations of APH induced an increase of macronuclear DNA content and cell size in non-dividing cells. In spite of the cell size increase, most proliferation of basal bodies, ciliogenesis and development of new oral primordia were prevented by the APH treatment. The division arrest induced by APH was partly overridden by caffeine (CAF) treatment, which caused the fragmentation ("pulverization") of the chromosomes in G2 micronuclei. Somatic progeny of dividers with pulverized micronuclei (APH+CAF strains) contained aneuploid and amicronucleate cells. The amicronucleate cells, after losing their oral structures and most of their cilia, and undergoing progressive disorganization of cortical structures, assumed an irregular shape ("crinkled") and were nonviable. "Crinkled" cells were not formed after APH + CAF treatment of the amicronuclear BI3840 strain, which contains some mic-specific sequences in its macronucleus. Most of the APH +CAF strains had a typical "*"- like conjugation phenotype: they did not produce pronuclei, but received them unilaterally from their mates and retained old macronuclei. However, 4 among 100 APH+CAF clones induced arrest at meiotic metaphase I in their wt mates. It is likely that the origin of such clones was enhanced by chromosome pulverization.     

Indispensable function of the germ nucleus for postconjugational stomatogenesis in Paramecium caudatum: Evidence against its genic function shown by hypohaploid micronuclei

It is known that the germinal micronucleus at the stages of gametogenesis and/or fertilization has an indispensable function for the postconjugational development of oral apparatus (stomatogenesis) in Paramecium caudatum. To determine whether this function is due to some specific genes in the micronucleus, postconjugational stomatogenesis was examined in the conjugation of haploid and hypohaploid cells. Haploid clones were obtained by conjugation between amicronucleate cells and diploid micronucleate cells. After conjugation between these haploid clones or between the haploid clones and amicronucleate clones, we succeeded in obtaining hypohaploid clones that have various types of nullisomic micronuclei. If a few genes in the micronucleus control postconjugational stomatogenesis, some hypohaploid micronuclei should undergo stomatogenesis normally, but others should not. In the present work, however, almost all the hypohaploid micronuclei developed the oral apparatus and formed food vacuoles. We can apparently rule out the possibility that a few specific genes of the micronucleus are required for postconjugational stomatogenesis in Paramecium caudatum, unless selection operates to retain the chromosomes with the essential gene(s). Dev. Genet. 23:142–150, 1998. © 1998 Wiley-Liss, Inc.     

Macronuclear differentiation in conjugating pairs of Tetrahymena treated with the antitubulin drug nocodazole

During Tetrahymena conjugation gamic nuclei (pronuclei) are produced, reciprocally exchanged, and fused in each mate. The synkaryon divides twice; the two anterior nuclei develop into new macronuclei while the two posterior nuclei become micronuclei. The postzygotic divisions were blocked with the antitubulin drug nocodazole (ND). Then pronuclei (gamic nuclei) developed directly into macronuclear anlagen (primordial macronuclei), inducing amicronucleate cells with two anlagen, or, rarely, cells with one anlagen and one micronucleus. ND had a similar effect on cells that passed the first postzygotic division inducing amicronucleate cells with two anlagen, while cells treated with ND at the synkarya stage produced only one large anlage. Different intracytoplasmic positioning of the nuclei treated with ND (pronuclei, synkarya and two products of the first division) shows that most of cell cytoplasm is competent for inducing macronuclear development. Only posteriorly positioned nuclei--products of the second postzygotic division--remain micronuclei. The total cell DNA content, measured cytophotometrically in control and in ND-induced amicronucleate conjugant cells with one and two anlagen, was similar in all three samples at 12 h of conjugation. Eventually, at 24 h this content was about 2 pg (8 C) per anlagen both in nonrefed control and in amicronucleate exconjugants. Therefore "large" nuclei developing in the presence of ND were true macronuclear anlagen.     

Genetic Analysis of Mating Type Differentiation in PARAMECIUM TETRAURELIA. II. Role of the Micronuclei in Mating-Type Determination

The two complementary mating types, O and E, of Paramecium tetraurelia are normally inherited cytoplasmically. This property has generally been interpreted to indicate the presence of cytoplasmic factors that determine macronuclear differentiation towards O or E. In these macronuclear-cytoplasmic interactions, the micronuclei were held to be unbiased and the determination to be established in the course of macronuclear development. In order to ascertain whether the micronuclei were actually neutral, amicronucleate clones were needed and a method to produce them was developed. In crosses between amicronucleate clones and normal micronucleate clones, we have observed regular deviations from cytoplasmic inheritance: the commonest deviation is that most O amicronucleate cells become E when they receive a micronucleus from an E partner. The data can be interpreted by assuming that the micronuclei are predetermined and that the apparent "cytoplasmic" inheritance of the two mating types is due, in E cells, to E-determining factors present in the cytoplasm and in the nucleus; and, in O cells, to O-determining factors present only or mainly in the nucleus.     

冠突伪尾柱虫有性生殖期间皮膜发育的核控制

通过显微手术去小核建立多个冠突伪尾柱虫（Pseudourostyla cristata)无小细胞系,并诱导它们与有小核细胞进行接合生殖,以评估小核及其衍生的大核原基在有性生殖期间对皮膜形态发生的影响,当无小核接合体从有小核配偶获得1枚配子核后,接合双方不仅能平行地继续核器演化,而且使第1次皮膜改组能够同步进行和正常发育,说明小核在有性周期中除了生殖功能外仍保留着某些控制皮膜发育的体功能,虽然大部分接合后体的大核原基在DNA贫乏期停止发育,但少数接合后体能够超越这一时期,并启动第2次皮膜改组和顺利完成其后续的有性发育全程,表明指令发动第2次皮膜发育的信号来自DNA贫乏期后以排出一核物质团块为标志的大核原基。     

棘尾虫无小核镜像骈体的获得及其生殖行为的实验观察

用在分裂期进行显微手术的方法和在陈旧培养中诱导的方法,从贻贝棘尾虫天然无小核系S_(10)中获得了无小核镜像骈体。对骈体的各种生殖行为进行了观察,并用孚尔根和改进的黑色素染色技术揭示了它们的细胞学细节。发现在本文描述的无小核骈体和过去报告的有小核骈体之间,在二分裂和生理再生上没有显著区别。无小核骈体和正常单体S_7之间的接合命运则是多样化的。在630个这样的接合对中,有257对发生了真正的接合并形成接合后体;有153对接合不久包囊化;在其余的220对中,一个接合体吸收了另一个,变成营养型的单体和有小核的骈体。前者占总吸收者的96.8％,后者只占3.2％。对接合和吸收两种情况的细胞学事件进行了详细观察。无小核骈体中的两组细胞质和双套大核能吸引其配偶中的小核,形成自己新的双套大核胚基及小核胚基,在本文的讨论中受到充分注意。     

Analysis of germinal aging in Paramecium caudatum by micronuclear transplantation

The role of the micronucleus in the age-dependent increase in mortality after conjugation in Paramecium has been investigated using micronuclear transplantation. The clone of Paramecium caudatum used for this study had a lifespan of about 750 fissions. In this clone, the fission rate began to decrease about 450 fissions after conjugation. Mortality after selfing conjugation also began to appear at about 450 fissions and gradually increased with clonal age. Cells at about 650 fissions showed 10–70% survival after selfing conjugation but when their micronuclei were transplanted into amicronucleate cells of about 450 fissions, the progeny survival increased to 70–90%. When micronuclei from cells 700–750 fissions old were transplanted into amicronucleate cells of 100–150 fissions, however, increase in progeny survival was very rare. The results indicate that micronuclei in cells up to the age of 650 fissions can function normally if the cytoplasmic environment is young.     

Holospora elegans-Infected Micronuclei of Paramecium caudatum Still Serve a Function During Vegetative Life1

The fission rate of Paramecium caudatum cells infected with the micronucleus-specific bacterium Holospora elegans was examined before and after elimination of the micronucleus. Uninfected cells, micronucleate and amicronucleate, were used as controls. Emicronucleation of Holospora elegans-infected cells causes a decrease of fission rates, as is observed after emicronucleation of uninfected cells. This is taken as an argument that infected micronuclei still serve a function for the vegetative life of P. caudatum.     

Disturbance of the determination of germinal and somatic nuclei by heat shock in Paramecium caudatum

During conjugation of Paramecium caudatum, nuclear determination occurs soon after the third postzygotic division: one of the four anterior nuclei becomes the micronucleus and the remaining three degenerate, while four posterior nuclei differentiate into macronuclear anlagen. Macronuclear differentiation is supposed to be dependent on a cytoplasmic differentiation factor. In this study, postzygotic cells were subjected to heat shock for 30 min and nuclear changes were observed by staining with carbol fuchsin solution. When heat shock was initiated during the period from metaphase to telophase of the third postzygotic division, cells showed an excess of macronuclear anlagen and were typically amicronucleate. Abnormal nuclear localization around the end of the third (last) postzygotic division may explain the origin of these kinds of cells. A similar phenomenon appeared after treatment with actinomycin D or emetine. Since heat shock did not inhibit macronuclear differentiation but destroyed the formation of micronuclei, some factor(s) probably plays an essential role in nuclear determination, especially in the protection of the micronuclei.     

Cytidine Analogues and Stomatogenic Recovery in Amicronucleate Paramecium tetraurelia and Paramecium jenningsi

Removal of the micronuclei of Paramecium tetraurelia and Paramecium jenningsi by micropipetting generates amicronucleate cell lines. These cell lines go through a period of growth depression for several dozen fissions, but they gradually recover. Amicronucleate cells in the depression period characteristically exhibit abnormal oral development, particularly reduction in the length of the buccal cavity and an abnormal pattern of the oral membranelles. To test the notion that the macronucleus is involved in the recovery of amicronucleate cell lines, DNA demethylation drugs were administered to amicronucleates in the depression period. After at least 4 fissions, the treated amicronucleates were assessed for their progress in recovery by scoring the proportion of cells with normal oral membranelles. Cvtidine analogues which demethylate cytosine specifically at the 5 position, namely 5-azacytidine, 5-aza-2'- deoxycytidine and 5-fluoro-2'-deoxycytidine. promoted recovery of the amicronucleates. Cytidine, 6-azacytidine, 2'-fluoro-2'-deoxy-cytidine and cytosine-β-D-arabinofuranoside did not. These results suggest that (i) 5-methylcytosine is present in the macronucleus of these Paramecium species, probably in small amounts and (ii) recovery of amicronucleates involves demethylation of macronuclear DNA. This implies that in normal cells the micronuclei are involved in maintaining the macronuclear DNA in a methylated state and hence the inactivation of the macronuclear sequences that are to be employed for stomatogenic recovery. A general mechanism for the control of gene expression may therefore be employed for the regulation of specific sequences.     

Proliferation and differentiation of chick skeletal muscle cells cultured in a chemically defined medium

By using the technique of nuclear transplantation in Paramecium [1], amicronucleate and renucleate clones were prepared in P. caudatum. The major differences between amicronucleate and micronucleate cells in the vegetative stage are elongation of cell cycle time, decrease in food vacuole formation, and shortening of the buccal cavity in the amicronucleate cells. These characteristics of amicronucleate cells are closely related with the absence of micronucleus, because all of these abnormalities were cured when the micronucleus was transplanted again into the amicronucleate. It is evident that the germinal micronucleus plays an important role not only during the sexual cycle but also in vegetative growth. Elongation of the cell cycle time in amicronucleates was also observed in P. bursaria and P. jenningsi.     

A Transmissible developmental block in Tetrahymena thermophila

When the amicronucleate mutant BI3840 of Tetrahymena thermophila is mated with normal micronucleate cells, it receives a pronucleus from its partner but there is no further nuclear development and the conjugants separate, retaining their original macronuclei. Both of these sexually mature exconjugants and any cells with which they are mated show an unconditional block in macronuclear development. Although prezygotic nuclear divisions, nuclear transfer and post-zygotic nuclear divisions appeared normal upon cytological analysis of Giemsa-stained conjugants, macronuclear development was invariably aborted. Since the original macronucleus was resorbed, the cells were rendered amacronucleate and they died. When no macronuclear development was initiated, as in crosses with the aneuploid strain A* (III), the exconjugants were viable and retained their original macronuclei. This pattern was invariant with three different strains serving as the original micronuclear source, and in the case of one non-BI3840 exconjugant, persisted for over 200 cell generations. Exconjugants from a cross of one of the micronuclear donors with strain A* (III) did not show arrested development when crossed. It thus appears likely that there is conjugal transfer of non-nuclear information originating in BI3840 which is self-replicating and which causes an arrest in macronuclear development.     

Nuclear Differentiation in Paramecium caudatum: Analysis by the Monoclonal Antibody against a Micronuclear Antigen

I obtained the monoclonal antibody 93A against a micronuclear antigen of the ciliate Paramecium caudatum . Immunocytochemical observations showed that the antigen 93A appeared in some portion of the micronucleus in every stage of life cycle. In dividing micronuclei, the antigen appeared mainly in their both poles and in fibrous structures between the poles. These results suggest that the micronuclear antigen 93A may be a component of microtubule organizing center and spindles. During nuclear differentiation in P. caudatum , four among eight postzygotic micronuclei differentiate new macronuclear anlagen and one becomes a new micronucleus and the remaining three degenerate. The micronuclear antigen 93A appeared in all of the eight nuclei in the early stage of macronuclear differentiation but then disappeared in the four macronuclear anlagen and eventually persisted only in the new micronucleus, showing that the newly developing macronuclear anlagen lose the micronuclear antigen 93A during their differentiation.     

Induced Resistance to 6-Methylpurine and Cycloheximide in Tetrahymena. II. Variation within Vegetative Cultures of Micronucleate T. THERMOPHILA and Amicronucleate T. PYRIFORMIS

Resistance to 6-methylpurine and cycloheximide has been induced in both the micronucleate species Tetrahymena thermophila and the amicronucleate species T. pyriformis. Resistance follows only after mutagen treatment and vegetative growth. The frequencies with which resistant variants are induced and the independence of mutagenesis and selection are demonstrated. All evidence is consistent with the hypothesis that macronuclear subunits are assorting in both species during vegetative growth to produce new phenotypes among subclones.     

Nuclear Differentiation in Exconjugants of Paramecium caudatum: Role of Nuclear Division in Differetiation

It has been known that, immediately after the third division of fertilization nucleus (synkaryon), nuclei localized near the posterior region of exconjugant are to be macronuclear anlagen and those near the anterior region are to be presumptive micronuclei in Paramecium caudatum. One of such posterior nuclei was transplanted into amicronucleate cell at vegetative phase in this work. The implanted nuclei were able to divide at every fission. Their DNA content was nearly equal to or less than ordinary micronuclei during vegetative phase. When conjugation was induced between clones obtained and amicronucleates, macronuclear anlagen developed from the division products of implanted nuclei and thereafter derivative caryonides were true to the marker gene of implanted nuclei. The results indicate that there was no intrinsic difference between nuclei localized anteriorly and those situated posteriorly in exconjugant. Differentiation of nuclei into macronucleus may be irreversible at the stage of anteroposterior localization of the nuclei. The role of nuclear division in differentiation may be only to transport the daughter nuclei into the cytoplasm/cortex differentiated anteroposteriorly.     

Nuclear functions in postconjugational development of Paramecium caudatum: Ability to form food vacuoles analyzed by nuclear elimination and implantation

Paramecium caudatum loses the ability to form food vacuoles at the crescent stage of the micronucleus from 5 to 6 hr after the initiation of conjugation and regains it immediately after the third division of the zygotic nucleus. To assess the micronuclear function in the development of the oral apparatus after coniugation, prezygotic micronuclei was removed from cells at various stages of conjugation, and their ability to form food vacuoles were examined. (1) When all of the prezygotic micronuclear derivatives were eliminated before the stage of formation of the zygotic nucleus, the exconjugant did not regain its ability. (2) When a zygotic nucleus or postzygotic nuclei were removed, in some cases the cell formed as many food vacuoles as did nonoperated cells after conjugation, while in other operated cells the number of food vacuoles was subnormal. (3) When a micronucleus from a cell at vegetative phase (G1) was transplanted into a cell of an amicronucleate mating pair at the stage between 8 and 9 hr after the initiation of conjugation, the implanted cell regained the ability to form food vacuoles. However, no cell regained the ability when the implantation was carried out within 1 hr after the separation of the mates. The results show that the micronucleus plays an indispensable role in the development of the oral apparatus at the stages of exchange of gametic nuclei and fertilization and that the micronucleus transplanted from asexual cells can fulfill this function. On the other hand, removal of the macronucleus from exconjugants showed that the maternal macronucleus also has an indispensable function in regaining the ability to form food Vacuoles. © 1992 Wiley-Liss, Inc.     

Histone rearrangements accompany nuclear differentiation and dedifferentiation in Tetrahymena

Histone synthesis and deposition into specific classes of nuclei has been investigated in starved and conjugating Tetrahymena. During starvation and early stages of conjugation (between 0 and 5 hr after opposite mating types are mixed), micronuclei selectively lose preexisting micronuclear-specific histones α, β, γ, and H3^F. Of these histones, only α appears to accumulate in micronuclear chromatin through active synthesis and deposition during the mating process. Curiously, α is not observed (by stain or label) in young macronuclear anlagen (4C, 10 hr of conjugation). Thus, young macronuclear anlagen are missing all of the histones which are known to be specific to micronuclei of vegetative cells. By 14–16 hr of conjugation, we observe active synthesis and deposition of macronuclear-specific histones, hv1, hv2, and H1, into new macronuclear anlagen (8C). Thus macronuclear differentiation seems well underway by this time of conjugation. It is also in this time period (14–16 hr) that we first detect significant amounts of micronuclear-specific H1-like polypeptides β and γ in micronuclear extracts. These polypeptides do not seem to be synthesized during this period, which suggests that β and γ are derived from a precursor molecule(s). Since these micronuclear-specific histones do not appear in micronuclear chromatin until after other micronuclei have been selected to differentiate as macronuclei, we suspect that micronuclear differentiation is also an important process which occurs in 10–16 hr mating cells. Our results also suggest that proteolytic processing of micronuclear H3^S into H3^F (which occurs in a cell cycle dependent fashion during vegetative growth) is not operative during most if not all of conjugation. Thus micronuclei of mating cells contain only H3^S which also seems consistent with the fact that some micronuclei differentiate into new macronuclei (micronuclear H3^S is indistinguishable from macronuclear H3). Interestingly, the only H3 synthesized and deposited into the former macronucleus of mating cells is the relatively minor macronuclear-specific H3-like variant, hv2. These results demonstrate that significant histone rearrangements occur during conjugation in Tetrahymena in a manner consistent with the fact that during conjugation some micronuclei eventually differentiate into new macronuclei. Our results suggest that selective synthesis and deposition of specific histones (and histone variants) plays an important role in the nuclear differentiation process in Tetrahymena. The disappearance of specific histones also raises the possibility that developmentally regulated proteolytic processing of specific histones plays an important (and previously unsuspected) role in this system.