5-Azacytidine affects the programming of expression of the somatic nucleus of Paramecium

This report introduces a new system in the study of programming of genomic function during development of the somatic nucleus of Paramecium tetraurelia. Previous works have established a definite, but replaceable, role of the germ nuclei (micronuclei) in oral development in the asexual cycle; their removal from the cell generates viable amicronucleate cell lines, which characteristically suffer a transient period of growth depression marked by abnormal oral development. Such cell lines gradually recover, showing that a compensatory mechanism is activated in the absence of the germ nuclei to bring the cell back to near-normal. To test the notion that the somatic nucleus (macronucleus) is involved in this compensation, cells possessing micronuclei were treated with 5-azacytidine during sexual reproduction when new somatic nuclei develop. These cells were then propagated asexually for a number of fissions in the absence of the drug, and thereafter micronuclei were removed from them. The amicronucleate cell lines generated in this manner clearly did not suffer a depression as severe as the untreated controls did in terms of growth rate and oral development, and they recovered much sooner. This supports the notion that the somatic nucleus is the physical basis of the compensatory mechanism. This study suggests that the stomatogenic sequences in question normally become repressed in the somatic nucleus developing in sexual reproduction, and that 5-azacytidine administered to the cells at this time could alter this programme which then persists during subsequent asexual propagation. The possibility that the somatic nucleus is programmed by methylation of cytosine at the 5' position is discussed.     

The Somatic Function of the Germ Nucleus in Pseudourostyla cris tat a: Asexual Reproduction and Stomatogenesis

The micronuclei of the ciliated protozoan Pseudourostyla cristata were eliminated by amputation shortly before binary fusion. The amicronucleate cell lines derived from regenerants were maintained for more than a year. They exhibited a lower viability and reduced vigor in asexual propagation. There was some improvement in the growth of the cell lines 1 mo after operation, but the growth rate remained subnormal even up to 1 yr of culture. The exact cause of the poor growth and survival in the first 3 wk after operation, whether the loss of the micronucleus or operational damage, remains to be determined. It is nevertheless clear that the micronucleus is important for subsequent asexual propagation. The amicronucleate cell lines were permanently crippled in morphogenesis, unlike the situation in Paramecium amicronucleates in which stomatogenesis returned to near-normal during asexual propagation. They always included some cells with a characteristically defective adoral zone of membranelles, reduced number of frontal-ventral-transverse cirri, and reduced body length. They were also reluctant to encyst. It is evident that the micronucleus is important for maintaining normality of the oral apparatus. It is postulated that the permanent stomatogenic crippling of amicronucleates might be related to genomic reduction in the developing macronucleus in sexual reproduction, as exhibited by other hypotrichs. The morphological defects associated with the adoral zone of membranelles may be rationalized as arising from the spreading of a zone of degeneration in the cortex affecting the left edge of the membranelles.     

Persistence of Oral Structures in the Sexual Process of Amicronucleate Paramecium tetraurelia

ABSTRACT In the sexual process, amicronucleate Paramecium tetraurelia , unlike micronucleates, fail to produce an oral apparatus, but resorb the pre-existing one. Exceptions were found in some amicronucleate cell lines in which about 1% of the cells possessed oral structures, including pieces of oral membranelles, sometimes complete with buccal cavity, after autogamy or conjugation. By following oral development in the sexual process in some detail, the present study supports the view that these oral structures are derived from the pre-existing oral apparatus and not newly developed from the oral primordium. The possible involvement of the micronucleus and the pre-existing oral apparatus in oral resorption is discussed. The possession of a functional oral apparatus after the sexual process may open up a new evolutionary avenue to the amicronucleates.     

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.     

瘦尾虫的小核在无性繁殖周期中对细胞形态结构的影响

通过显微切割建立瘦尾虫无小核细胞系,并与原细胞系及切割后再生的有小核细胞系进行对照观察。结果表明,无小核细胞的形态结构,尤其是口器出现高比率的畸形。在生长静止期,无小核细胞系群体中约23 % (36/155)的细胞完全失去了波动膜。与此同时,这些细胞的口围带也显出异常。一些无小核细胞的大核也出现异常,有的细胞仅含有1枚大核,有的则含有4枚,而不是通常的2枚;还有少数细胞的大核在非细胞分裂期进行分裂。上述结果提示,在无性繁殖周期中,瘦尾虫的小核对于维持正常的细胞形态结构、尤其是保持胞口结构的稳定性和大核数的恒定起着重要的作用[动物学报52 (2) : 383 -388 , 2006]。     

The somatic function of the micronucleus in asexual reproduction of Paramecium: thermosensitivity of amicronucleates

It has been established that following removal of the micronucleus in Paramecium tetraurelia, the amicronucleate cell line enters a depression period, characterized by slow growth rate and oral abnormalities, at normal growth temperature (27°C). Such cell lines gradually recover in growth rate and stomatogenesis. In the present study, 4 recovered amicronucleate cell lines were challenged with high temperatures (35°C, 36°C, and 36.5°C). They exhibited growth rate reduction and abortive cytokinesis at 35°C and 36°C, and died at 36.5°C. In addition, they demonstrated oral defects similar to those observed in the depression period: disruption of the regular oral membranellar pattern, reduction in the length of the oral apparatus, and impaired phagocytosis (food vacuole formation). These high temperature-induced abnormalities were largely restricted to amicronucleates, and were rare or seen to a much lesser extent in sister micronucleate cell lines. This study demonstrates the participation of the micronucleus in conferring thermotolerance on the cells. It is hypothesized that the micronucleus specifies heat-shock proteins to maintain the integrity of oral and somatic cytoskeletal elements at high temperature.     

念珠伪角毛虫小核体功能初步研究

为了研究念珠伪角毛虫的小核是否具有及具有怎样的体功能,采用显微切割手术去小核并建立无小核细胞系。经蛋白银染色鉴定,无小核细胞系群体中大多数细胞的形态结构存在缺陷：口围带的部分小膜缺损或排列紊乱,大核的数目和形态也不正常。这表明,念珠伪角毛虫的小核对于保持口围带结构的完整性以及大核的数目和形态结构的稳定性起着明确的作用。     

Interactions between newly developed macronuclei and maternal macronuclei in sexually immature multinucleate exconjugants of Paramecium caudatum

The macronucleus of Paramecium caudatum controls most cellular activities, including sexual immaturity after conjugation. Exconjugant cells have two macronuclear forms: (1) fragments of the maternal macronucleus, and (2) the new macronuclei that develop from the division products of a fertilization micronucleus. The fragments are distributed into daughter cells without nuclear division and persist for at least eight cell cycles after conjugation. Conjugation between heterokaryons revealed that the fragmented maternal macronuclei continued to express genetic information for up to eight cell cycles. When the newly developed macronucleus was removed artificially within four cell cycles after conjugation, the clones regenerated the macronuclear fragments (macronuclear regeneration; MR) and showed mating reactivity, because they were sexually mature. However, when the new macronucleus was removed during later stages, many MR clones did not show mating reactivity. In some extreme cases, immaturity continued for more than 50 fissions after conjugation, as seen with normal clones that had new macronuclei derived from a fertilization micronucleus. These results indicate that the immaturity determined by the new macronucleus is not annulled by the regenerated maternal macronucleus. Mature macronuclear fragments may be "reprogrammed" in the presence of the new macronucleus, resulting in their expression of "immaturity."     

Analysis of mating type determination by transplantation of O macronuclear karyoplasm in Paramecium tetraurelia

The odd (O) or even (E) mating type in Paramecium tetraurelia is determined during the first cell cycle after new macronuclear development. The present paper demonstrates that mating type E is irreversibly determined at the end of the first cell cycle. Direct evidence comes from transplanting O macronuclear karyoplasm containing O-determining factor into E autogamous cells during a new postzygotic macronuclear development. Transplantation of O macronuclear karyoplasm into E autogamous cells at 7–8 hr after the origin of the macronucleus from a product of the synkaryon produces nearly 100% O mating type among the exautogamous cell lines but almost none 10–11 hr after the origin of the macronucleus (around the end of the first cell cycle). The macronuclear anlagen at the stage at which mating type E seems to be fixed contains about 20 times as much DNA as the vegetative G1 micronucleus. The O-determining factor shifting E cells toward O mating type by transplanting O macronuclear karyoplasm is also produced by the newly developed macronucleus in an effective concentration at 10–11 hr after the sensitive period and produced at full levels by the third cell cycle. The level of O factor in the macronucleus then gradually declines with subsequent repeated rounds of DNA synthesis and is finally lost by the eighth cell cycle.     

Regulation of macronuclear DNA content in Paramecium tetraurelia

The macronucleus of Paramecium divides amitotically, and daughter macronuclei with different DNA contents are frequently produced. If no regulatory mechanism were present, the variance of macronuclear DNA content would increase continuously. Analysis of variance within cell lines shows that macronuclear DNA content is regulated so that a constant variance is maintained from one cell generation to the next. Variation in macronuclear DNA content is removed from the cell population by the regulatory mechanism at the same rate at which it is introduced through inequality of macronuclear division. Half of the variation in macronuclear DNA content introduced into the population at a particular fission by inequality of division is compensated for during the subsequent period of DNA synthesis. Half of the remaining variation is removed during each subsequent cell cycle. The amount of variation removed in one cell cycle is proportional to the postfission variation. The cell's power to regulate DNA content is substantially greater than that required to compensate for the small differences that arise during division of wild-type cells. For example, a constant variance was still maintained when the mean difference between sister cells was increased to ten times its normal level in a mutant strain. The observations are consistent with a replication model that assumes that each cell synthesizes an approximately constant amount of DNA which is independent of the initial DNA content of the macronucleus. It is suggested that the amount of DNA synthesized may be largely determined by the mass of the cell.     

Bacteriophage lambda DNA fragments replicate in the Paramecium macronucleus: absence of active copy number control.

We show that bacteriophage lambda DNA fragments microinjected into the macronucleus of the ciliated protozoan Paramecium can replicate as unit-length linear molecules. These linear DNA molecules are substrates for the addition of Paramecium telomeres by an endogenous telomerase. The linear DNA pieces can exist at copy numbers much higher than that of typical endogenous macronuclear chromosomes. We show that the copy number of injected DNA many fissions after microinjection reflects that of the original input copy number, suggesting that active control of copy number does not occur. Instead, the results suggest that injected DNA is replicated once per cell division.     

Elimination of the germ nuclei of Paramecium tetraurelia by laser microbeam irradiation

The germ nuclei (micronuclei) of Paramecium tetraurelia can be eliminated successfully by irradiating the micronucleus with an argon-ion laser microbeam after sensitization with the dye acridine orange. No immediate cytological damage of the irradiated micronuclei is visible, but they are lost before they enter the next division. This method produces cell lines lacking micronuclei (i.e., amicronucleates). These amicronucleates provide favorable materials for the study of micronuclear functions as well as intra-and inter-specific nucleocytoplasmic interactions. Some preliminary observations show that the micronucleus is not required for macronuclear fragmentation and macronuclear regeneration during sexual reproduction, but suggest that the micronucleus might participate in some asexual cellular function in addition to their gametic role.     

Abnormalities in Nuclear Behavior and Mating Type Determination in Cytoplasmically Bridged Exconjugants of Doublet Paramecium aurelia*

Exconjugant clones of Paramecium aurelia stock 51S, syngen 4, which fail to separate prior to the 1st fission have numerous cytologic and mating type determination anomalies. The doublets have abnormal distribution of macronuclear anlagen, fewer macronuclear fragments per cell, and abnormalities in numbers of micronuclei. Despite apparent cell fusion and mixture of cytoplasm, the singlets arising from each side of the doublet may be of opposite mating types, and mating type determination may remain unstable for 1 or more fissions in contrast to the usual pattern of mating type determination before the 1st postconjugation fission.     

Regulation of Macronuclear DNA Content in Paramecium tetraurelia*†

Synopsis.
The amitotic division of the macronucleus of Paramecium tetraurelia produces daughter macronuclei which frequently differ in DNA content. In wild-type cells these differences are small, but can be increased substantially by the action of mutant genes. The variance in macronuclear DNA content would increase continuously if there were no mechanism to regulate it. Paramecium has a very effective regulatory mechanism—all cells synthesize similar amounts of macronuclear DNA, regardless of the number of macronuclei or their prereplication DNA content. DNA synthesis is controlled at the level of macronuclear subunits, and the postreplication macronucleus consists of a mosaic of subunits that have undergone different numbers of replication events during the previous cell cycle. It is evident from experimental results that the amount of DNA synthesized can be influenced by the total size or mass of the cell. Experimental modification of the initial DNA content leads to no change in the amount of DNA synthesized, or in the subsequent protein content of the cells, but modification of cell size causes corresponding changes in the amount of DNA synthesized and in the size of the macronucleus. The implications of these observations for cell growth and the cell cycle are discussed.     

Trichocyst phenotype transformation induced by macronuclear transplantation in Paramecium tetraurelia

A portion of the macronucleus of wild-type cells of Paramecium tetraurelia was removed and was injected into cells homozygous for the ftA mutation. The ftA mutants make defective trichocysts and are unable to perform normal trichocyst exocytosis. After injection, approx. 30% of the surviving cells show a phenotype shift from mutant to wild-type. This shift is stable during subsequent vegetative growth until clonal death. If, however, the hybrid cell lines are brought to autogamy (which discards the existing macronucleus and forms a new one from sexual products derived from a micronucleus), then the lines revert to the ftA phenotype. Since micronuclei were not transplanted, the phenotypic reversion after autogamy is to be expected, and demonstrates that the transformation affects the macronucleus only. A second series of injections involved transfer of a portion of the macronucleus from cells homozygous for the trichocyst ptA mutation into ftA host cells. These two mutations are genetically complementary, so the injection should be genetically equivalent to forming a double heterozygote. Approx. 20% of the injection survivors shift to wild-type. This shift is also vegetatively stable unless autogamy occurs; after autogamy, reversion to the ftA phenotype is seen. These results show that a portion of a macronucleus can be successfully transplanted from one cell to another and that, in the host cytoplasmic environment, normal gene expression and replication of a transplanted macronucleus does occur. The technique of macronuclear transplantation is significant to studies of the macronuclear contribution to clonal aging, and to studies on genetic control over trichocyst development.     

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.     

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.     

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.     

Internuclear control of DNA synthesis in exconjugant cells of Paramecium caudatum

An exconjugant cell of Paramecium caudatum has two kinds of macronuclei, fragmented prezygotic macronuclei and postzygotic new macronuclei (anlagen). Although the DNA synthesis in the fragmented prezygotic macronucleus continues until the third cell cycle after conjugation, selective suppression of the DNA synthesis in the prezygotic macronucleus takes place at the fourth cell cycle. The inhibition of DNA synthesis in prezygotic fragmented macronuclei is due to the presence of a postzygotic macronucleus (anlage) in the same cytoplasm because the inhibition does not occur when the postzygotic macronucleus (anlage) is removed by micromanipulation during the third or fourth cell cycle. Well-developed postzygotic macronuclei (anlagen) with full ability to divide have the ability to depress the DNA synthesis of prezygotic macronuclear fragments. The suppression of DNA synthesis in prezygotic macronuclear fragments seems to be irreversible. Competition for the limited amount of DNA precursors also plays an important role in the onset of the selective suppression of the DNA synthesis.