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."     

Morphological apoptotic characteristics of the post-meiotic micronuclei in Paramecium caudatum

In a previous study, the apoptotic degeneration of meiotic products outside the paroral region of Paramecium caudatum was indirectly demonstrated by means of “apofluor” staining. In this experiment, conjugating pairs and exconjugants of P. caudatum were stained with either “apofluor” or carbol fuchsin or both to find some direct evidence to demonstrate the apoptotic characteristics of this process. As a result, asynchronous meiotic nuclear degeneration was observed. Furthermore, a number of additional meiotic nuclei were found. Disintegrating/dividing meiotic nuclei outside the paroral region were observed, which might be the origin of these additional meiotic nuclei. Condensed chromatin and disintegrated chromatin attached to the nuclear membrane were also observed in degenerating nuclei, which are the typical morphological characteristics of apoptosis. Comparison of the cells stained by the above two methods indicated that “apofluor”-stained meiotic nuclei could not be detected by carbol fuchsin in some cells, which suggests a time lag between meiotic nuclear DNA degradation and their eventual disappearance. In this study, some direct evidence was found to show that the meiotic nuclear degeneration in P. caudatum is of apoptotic nature, which further confirmed our previous study (Yang et al. 2007) and indicated that morphological apoptotic characteristics discovered in multicellular organisms do exist in unicellular eukaryotic ciliate protozoa.     

Behavior of germinal micronuclei under control of the somatic macronucleus during conjugation in Paramecium caudatum.

In conjugating pairs of Paramecium caudatum, the micronuclear events occur synchronously in both members of the pair. To find out whether micronuclear behavior is controlled by the somatic macronucleus or by the germinal micronucleus, and whether or not synchronization of micronuclear behavior is due to intercellular communication between conjugating cells, the behavior of the micronucleus was examined after removal of the macronuclei from either or both cells of a mating pair at various stages of conjugation. When macronuclei were removed from both cells of a pair, micronuclear development was arrested 1 to 1.5 hr after macronuclear removal. When the macronucleus of a micronucleate cell mating with an amicronucleate cell was removed later than 3 to 3.5 hr of conjugation, that is, an early stage of meiotic prophase of the micronucleus, micronuclear events occurred normally in the operated cell. These results suggest that most micronuclear events are under the control of the macronucleus and that the gene products provided by the macronucleus are transferable between mating cells. One such product is required for induction of micronuclear division and is provided just before metaphase of the first meiotic division of the micronucleus. This factor is effective at a lower concentration in the cytoplasm and/or is more transferable between mating cells than the factors required for other stages. This factor, which seems to be present at least until the stage of micronuclear disintegration, is able to induce repeated micronuclear division as long as it remains active. The factor can act on a micronucleus which has not passed through a meiotic prophase. Moreover, the results suggest the existence of a second factor which is provided by the macronucleus after the first meiotic division that inhibits further micronuclear division.     

Dynamic behaviour of stationary pronuclei during their positioning in Paramecium caudatum

During conjugation of Paramecium caudatum, there are two well-known stages when nuclear migration occurs. What happens to the nuclei is closely related to their localisations in cells. The first of these stages is the entrance of one meiotic product into the paroral region. This nucleus survives, while the remaining three outside this area degenerate. The second stage is the antero-posterior localisation of eight synkaryon division products. Four posterior nuclei are differentiated into macronuclear anlagen, whereas four anterior nuclei remain as the presumptive micronuclei. In this experiment, the process of the third prezygotic division of P. caudatum was studied with the help of protargol staining. Here, a third nuclear migration was discovered. By two spindle turnings and two spindle elongations, stationary pronuclei were positioned near migratory pronuclei. This positioning of stationary pronuclei could shorten the distance for transferred migratory pronuclei to recognise and reach the stationary pronuclei. This fosters the synkaryon formation of P. caudatum.     

Insights into Three Whole-Genome Duplications Gleaned from the Paramecium caudatum Genome Sequence

Paramecium has long been a model eukaryote. The sequence of the Paramecium tetraurelia genome reveals a history of three successive whole-genome duplications (WGDs), and the sequences of P. biaurelia and P. sexaurelia suggest that these WGDs are shared by all members of the aurelia species complex. Here, we present the genome sequence of P. caudatum, a species closely related to the P. aurelia species group. P. caudatum shares only the most ancient of the three WGDs with the aurelia complex. We found that P. caudatum maintains twice as many paralogs from this early event as the P. aurelia species, suggesting that post-WGD gene retention is influenced by subsequent WGDs and supporting the importance of selection for dosage in gene retention. The availability of P. caudatum as an outgroup allows an expanded analysis of the aurelia intermediate and recent WGD events. Both the Guanine+Cytosine (GC) content and the expression level of preduplication genes are significant predictors of duplicate retention. We find widespread asymmetrical evolution among aurelia paralogs, which is likely caused by gradual pseudogenization rather than by neofunctionalization. Finally, cases of divergent resolution of intermediate WGD duplicates between aurelia species implicate this process acts as an ongoing reinforcement mechanism of reproductive isolation long after a WGD event.     

Effects of perfluorinated amphiphiles on backward swimming in Paramecium caudatum

PFOS and PFOA are ubiquitous contaminants in the environment. We investigated the effects of fluorochemicals on calcium currents in Paramecium caudatum using its behavioral changes. Negatively charged amphiphiles prolonged backward swimming (BWS) of Paramecium. PFOS significantly prolonged BWS, while PFOA was less potent (EC(50): 29.8+/-4.1 and 424.1+/-124.0microM, respectively). The BWS prolongation was blocked by cadmium, indicating that the cellular calcium conductance had been modified. The positively charged amphiphile FOSAPrTMA shortened BWS (EC(50): 19.1+/-17.3). Nonionic amphiphiles did not affect BWS. The longer-chain perfluorinated carboxylates PFNA and PFDA were more potent than PFOA (EC(50): 98.7+/-20.1 and 60.4+/-10.1microM, respectively). However, 1,8-perfluorooctanedioic acid and 1,10-perfluorodecanedioic acid did not prolong BWS. The critical micelle concentration (CMC) and BWS prolongation for negatively charged amphiphiles showed a clear correlation (r(2)=0.8008, p<0.001). In summary, several perfluorochemicals and PFOS and PFOA had similar effects in Paramecium, while chain length, CMC, and electric charge were major determinants of BWS duration.     

Local Degeneration of Cilia and Nuclear Activation during Sexual Reproduction in Paramecium caudatum

The correlation between local degeneration of cilia and nuclear activation during chemically induced autogamy in Paramecium caudatum was studied by experimental interruption of autogamy. When the induction of autogamy was interrupted 60 min after the onset of chemical induction, nuclear activation did not occur and ciliary regeneration took place at the deciliated surface. On the contrary, when it was interrupted 3 hr after the onset of induction, nuclear activation was not stopped and no reciliation was observed. At this critical stage, the deciliated zone was seen to extend to the right wall of the gullet. These findings suggest that there is a transition point in activation processes. It is conceivable that in autogamous cells that have gone beyond this transition point all processes of nuclear activation proceed, and that the ability to regenerate cilia is lost by the end of autogamy, even if the stimulus for autogamy is removed.     

Nuclear differentiation in Paramecium tetraurelia: Transplantation of vegetative micronuclei into early exconjugants

The micronucleus of Paramecium tetraurelia normally gives rise to the macronucleus by going through the series of nuclear events of meiosis, mitosis and fusion during sexual reproduction. By transplanting the micronucleus from vegetative cells into early exconjugants, we have persuaded the micronucleus to take a short cut to differentiate directly into a macronucleus. This demonstrates that the course of development of the micronucleus is flexible and can be altered by stage-specific cytoplasmic factors.     

Cd++对草履虫种群的毒性作用

通过研究20℃培养条件下Cd 对草履虫(Paramecium caudatum)种群毒性影响,结果表明:Cd 对草履虫的24hLC50为0.3294mg/L,Cd 浓度对草履虫的种群增长率有极显著影响,多重比较结果显示高浓度组(0.25和0.30mg/L)下草履虫的种群增长率显著小于低浓度下(0.05、0.15和0.20mg/L)的值。回归分析结果表明草履虫种群增长率(Y,/d)与Cd 浓度(X,mg/L)之间呈曲线相关。在一定浓度范围内,草履虫种群增长率随Cd 浓度的升高而呈增大,但高的Cd 浓度显著地降低了草履虫的种群增长率;当Cd 浓度为0.1179mg/L时,草履虫种群有最大增长率1.5442/d。各浓度组与对照组之间的t-检验结果表明在较低的Cd 浓度范围内(0.05、0.10和0.15mg/L)草履虫的种群增长率显著大于对照组的值,而Cd 浓度为0.30mg/L时的草履虫种群增长率显著小于对照组的值。     

Autonomous replication and addition of telomerelike sequences to DNA microinjected into Paramecium tetraurelia macronuclei.

Paramecium tetraurelia can be transformed by microinjection of cloned serotype A gene sequences into the macronucleus. Transformants are detected by their ability to express serotype A surface antigen from the injected templates. After injection, the DNA is converted from a supercoiled form to a linear form by cleavage at nonrandom sites. The linear form appears to replicate autonomously as a unit-length molecule and is present in transformants at high copy number. The injected DNA is further processed by the addition of paramecium-type telomeric sequences to the termini of the linear DNA. To examine the fate of injected linear DNA molecules, plasmid pSA14SB DNA containing the A gene was cleaved into two linear pieces, a 14-kilobase (kb) piece containing the A gene and flanking sequences and a 2.2-kb piece consisting of the procaryotic vector. In transformants expressing the A gene, we observed that two linear DNA species were present which correspond to the two species injected. Both species had Paramecium telomerelike sequences added to their termini. For the 2.2-kb DNA, we show that the site of addition of the telomerelike sequences is directly at one terminus and within one nucleotide of the other terminus. These results indicate that injected procaryotic DNA is capable of autonomous replication in Paramecium macronuclei and that telomeric addition in the macronucleus does not require specific recognition sequences.     

Isolation and characterization of the hemoglobin from Paramecium caudatum

• 1.1. The hemoglobin of Paramecium caudatum has been purified to a state of homogeniety on polyacrylamide disc electrophoresis.
• 2.2. The hemoglobin appears as a single molecular species with a mol. wt of 13,500 daltons as determined by SDS disc electrophoresis.
• 3.3. An isoelectric point of 4.27 was calculated from isoelectric focusing experiments using ampholines in a pH range of 3–6.
• 4.4. The amino acid composition is: lys₅, his₂, arg₃, asp₁₂, thr₁₁, ser₉, glu₁₆, pro₂, gly₁₈, ala₂₁, cys₀, val₁₂, met₂, ile₂, leu₈, tyr₂, phe₆.
• 5.5. The spectra of three ferrous and the ferric cyanmethemoglobin derivatives are presented.

     

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.     

Developmental expression of macronuclear specific antigen in Paramecium caudatum

We obtained a monoclonal antibody (MA-1) specific for macronuclei of the ciliate Paramecium caudotum and P. dubosqui. Immunoblotting showed that the antigen was a poly-peptide of 50 kilodalton (kDa). During the process of nuclear differentiation in P. caudatum, the MA-1 antigens appeared in the macronuclear anlagen immediately after four out of eight post zygotic nuclei differentiated morphologically into the macro-nuclear anlagen. Afterwards, the antigens could be detected in the macronucleus through the cell cycle, and disappeared when the macronucleus began to degenerate in exconjugant cells. These results suggest that the antigens may play a role in the differentiation and function of the macronucleus. © 1992 Wiley-Liss, Inc.