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。     

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]。     

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。     

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.     

Reorganization in Amicronucleates with Defective Mouth of the Ciliate Pseudourostyla levis1

ABSTRACT. The purpose of this work is to examine the reorganization process in amicronucleates with defective mouth of the multimicronuclear ciliate, Pseudourostyla levis. The amicronucleates were derived from fragments obtained by transection of normal micronucleates. The cell size of the amicronucleates was extremely unstable and varied from 57.5 to 276.3 μm long (n = 146), whereas the micronucleates kept rather stable cell lengths with a range from 162.5 to 266.3 μm (mean ± SD = 213.1 ± 19.6 μm, n = 206). The renucleates obtained by transplantation of a micronucleus to an amicronucleate returned to almost normal cell size (mean ± SD = 203.7 ± 16.5 μm long, n = 54). Under the usual culture conditions in the amicronucleate cell line, the number of abnormal cells with defective mouth rapidly increased, up to about 60%, until a stationary phase. Similarly, abnormal cells also appeared in micronucleates although the frequency was always less than 10%. The mean number of membranelles in the normal adoral zone of membranelles (AZM) was 82.6 ± 3.9 (±SD, n = 49) vs. 57.3 ± 7.9 (n = 81) in ciliates with defective mouths. The missing part of the AZM was always the anterior part of the lapel. Cells with defective mouth underwent reorganization (= physiological regeneration) under the usual culture conditions. During reorganization, the lapel part of the old AZM was transformed into a new collar part. The defective mouth was repaired through this developmental process. These results suggest that in P. levis the decrease of food supply often leads to the loss of a specific part of the AZM and that this membranellar loss is suppressed by the existence of 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.     

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.     

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.     

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.     

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.     

阔口尖毛虫皮层纤毛器微管在不同生理条件下的分化

应用激光扫描共聚焦显微术,显示腹毛类纤毛虫阔口尖毛虫(Oxytricha platystoma)无性生殖过程中,新的口围带、波动膜、额腹横棘毛、左右缘棘毛微管先后分化,老纤毛器微管去分化,细胞分裂产生各含一套纤毛器微管的前、后两仔虫;生理改组过程中,口围带、波动膜、额腹横棘毛、左右缘棘毛微管发生去分化和再分化,细胞皮层微管胞器更新形成含一套纤毛器微管的新细胞。结果表明阔口尖毛虫在无性生殖和生理改组这两种不同的生理条件下,其纤毛器微管结构的形成或更新可能具有相同的细胞调控机制,形态发生中老纤毛器结构可能对新结构的发生和发育具有诱导定位和物质贡献的作用。     

Redescription of the rare heterotrichid ciliate,Copemetopus subsalsus Villeneuve-Brachon, 1940

The morphology and infraciliature of the rare heterotrichid ciliate Copemetopus subsalsus Villeneuve-Brachon, 1940 were studied in live and protargol-impregnated specimens and also by scanning electron microscopy. It is characterized by a dumbbell-shaped macronucleus, a specific dorsal brush cilia, an oblique peristome, and a distinct S-shaped adoral zone of membranelles equipped with whip-shaped anterior adoral membranelles.     

Nuclear transplant studies of the determination of mating type in germ nuclei of Paramecium tetraurelia

The micronucleus from vegetative cells of one mating type (O or E) in Paramecium tetraurelia was transplanted by micropipet into amicronucleate cells of opposite mating type (E or O). When autogamy was induced in the recipient cells, they developed new macronuclei and micronuclei derived from the transplanted micronucleus and usually expressed the same mating type as the recipients. The results indicate that micronuclei in the asexual phase may be undetermined for mating type. Recipient E cells in which the macronucleus had been previously removed were transplanted with a whole macronucleus from an O cell. Their mating type was soon transformed E to O before the occurrence of autogamy, and remained O after autogamy. This demonstrates that the transplanted macronucleus determined the O cytoplasmic state to determine the developing zygotic macronucleus for mating type O. It is unlikely that the micronucleus is determined for mating type in O or E cell during the asexual cycle.     

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.     

Morphological and molecular characterization of Aspidisca hongkongensis spec. nov. (Ciliophora,Euplotida) from the South China Sea

The morphology and infraciliature of a new ciliate, Aspidisca hongkongensis spec. nov., a large marine form isolated from the coastal waters near Hong Kong, were investigated. It differs from the congeners by the combination of cell size (about 90×60 μm in vivo), 9–11 conspicuous dorsal ridges, one fine and seven strong frontoventral cirri, six membranelles in anterior portion of adoral zone, and 17–23 membranelles in posterior portion of adoral zone. The comparison with similar congeners clearly supports the distinction of this new species based on morphological and small subunit (SSU) rRNA gene sequence studies. The SSU rRNA gene sequence similarity within the genus is about 75.3–92.1% which indicates that this is a distinct form. Phylogenetic trees based on SSU rRNA gene information show that A. hongkongensis groups with A. leptaspis.     

Physiological Reorganization and Post-Traumatic Regeneration in Stylonychia mytilus: Reflections on Developmental Constraints and the Evolutionary Origin of Alternative Modes of Asexual Morphogenesis

We investigated development of cortical ciliature in Stylonychia mytilus during starvation-induced physiological reorganization, and during regeneration following amputation of the anterior part of the cell. Cortical reorganization in the two processes is generally similar. The posterior part of the adoral zone of membranelles is resorbed and replaced with newly assembled membranelles. The pre-existing set of ventral cirri and dorsal bristles is entirely resorbed and replaced with new ones. Regenerants exhibit posterior displacement of the frontal-ventral-transverse cirri primordium and the undulating membrane primordium, and recruit basal bodies from ectopic locations for the development of these ciliature. This illustrates flexibility in the initiation site of ciliary primordia, and opportunism in utilizing building blocks. Such morphogenetic versatility of hypotrichs provides the basis for the operation of a global control of pattern formation, which governs cortical reorganization in dividers, and additionally, in the absence of the prerequisites for binary fission, alternative modes of cortical development such as physiological reorganization or regeneration. These considerations suggest that the three processes are homologous and that physiological reorganization and regeneration have evolved from binary fission. In physiological reorganization and regeneration, the micro- and macronuclei reorganize to resemble that in binary fission; these nuclear events are considered evolutionary relics of the nuclear development of binary fission. Tetrahymena also exhibits such morphogenetic flexibility; stomatogenesis is under global control, so that asexual cells can replace its oral apparatus without undergoing binary fission. Paramecium , on the other hand, adopts a more rigid strategy in relying heavily on pre-existing structures for morphogenetic cues; this could have imposed constraints in the exploration of alternative modes of asexual development.     

Defective Micronuclei and Genomic Exclusion in Selected C* Subclones of Tetrahymena1

SYNOPSIS. C* is a structurally heterogeneous clone containing normal and crinkled cells which are amicronucleate. Selection for structurally normal subclones did not result in the genesis of a diploid cell line. The stem cell of C* appears to be aneuploid with about 4 chromosomes instead of the usual 10, and from this line cells with fewer chromosomes or without micronuclei are generated. Only cells containing a portion of the micronucleus are viable. Amicronucleate cells die. They also mate very poorly. Consequently, little difference is observed between subclones having different proportions of amicronucleate cells in their ability to generate viable offspring by genomic exclusion. Our observations are discussed in terms of the separate functions of micronucleus and macronucleus and ways in which the nuclei seem to interact.     

阔口尖毛虫无性生殖和生理改组过程的比较

阔口尖毛虫无性生殖中先后发生后口围带原基,前、后的波动膜原基,额腹横棘毛原基和左、右缘棘毛原基,老口围带也在此期间更新,结果形成2套新纤毛结构,原老结构瓦解消失;生理改组时按同样顺序产生口围带原基等几类腹面纤毛原基,结果形成1套新纤毛结构,替换老结构。在这两个截然不同的过程中,新纤毛结构的分化和老纤毛结构退化时也表现出某些相似的特征。作者据此推测,这种纤毛虫无性生殖和生理改组中,纤毛原基的发生、发育和定位在细胞控制机理上可能是相同的。     

Morphologic and molecular data suggest that Lynnella semiglobulosa n. g., n. sp. represents a new family within the subclass Choreotrichia (Ciliophora, Spirotrichea)

The morphology, morphogenesis, and phylogeny of an undescribed oligotrich sensu lato (s. lat.) ciliate, Lynnella semiglobulosa n. g., n. sp., found in Daya Bay, southern China, were investigated. This species shares some features with both oligotrichs sensu stricto and choreotrichs, but most morphological and morphogenetic characters as well as the phylogenetic analysis suggest that it should be assigned into subclass Choreotrichia temporarily. Lynnella semiglobulosa is distinguished from members of all known genera and families of the subclass Choreotrichia by a unique combination of characteristics of the buccal and somatic ciliatures. Thus, a new family Lynnellidae n. fam. and a new genus Lynnella n. g. are proposed for it. The new family is distinguished by an open adoral zone of membranelles (AZM) in which, however, there are no ventral membranelles; the distal and proximal portions of the new adoral zone lie close to each other forming an open circle in stomatogenesis. The new genus Lynnella is characterized by possessing two longitudinally oriented somatic kineties, one dorsal and one ventral, several proximal membranelles progressively lengthened toward the proximal end of adoral zone, and two macronuclear nodules. In phylogenetic analyses based on small subunit rRNA gene sequences, L. semiglobulosa clustered basally to all choreotrichs, but with relatively weak support; nevertheless, the possibility of a relationship with the subclass Oligotrichia was not rejected by the approximately unbiased nor Shimodaira-Hasegawa test. Based on morphological, morphogenetic, and molecular evidence of L. semiglobulosa, it is confirmed that the open AZM should be a plesiomorphic character of oligotrichs s. lat. as suggested previously.