Kazuyuki Mikami

Phylogenetic relationships between Vorticella convallaria and other species inferred from small subunit rRNA gene sequences.

Abstract Vorticellid ciliates generally dwell in freshwater. In nature, the species have up until now been identified by comparison with previous descriptions. It is difficult to identify between species of the genus Vorticella, because the morphological markers of vorticellid ciliates described in reports are limited and variable. Unfortunately, culturing them has only succeeded with certain species such as Vorticella convallaria, but many others have been impossible to culture. To find out whether the sequence of a small subunit rRNA gene was an appropriate marker to identify vorticellid ciliates, the gene was aligned and compared. Finding a new convenient method will contribute to research on vorticellid ciliates. In strains of V. convallaria, classified morphologically, some varieties of the SSrRNA gene sequences were recognized, but there were large variations within the same species. According to the phylogenetic tree, these strains are closely related. However, the difference was not as big as between Vorticella and Carchesium. In addition, Carchesium constructed a distinct clade from the genus Vorticella and Epistylis. These results show the possibility that the SSrRNA gene is one of the important markers to identify species of Vorticella. This study is first to approach and clarify the complicated taxa in the genus Vorticella.

Macronuclear development and gene expression in the exconjugants of Paramecium caudatum

Abstract Postzygotic nuclei are determined to be macronuclear anlagen immediately after the third division of the fertilization nucleus in Paramecium caudatum. Soon after determination, chromatic aggregates appear in the anlagen. Microspectrophotometry revealed that a marked increase in the DNA content of macronuclear anlage occurred after disintegration of the chromatic aggregates, about 18 hr after nuclear determination. The phenotypic expression of five genes was investigated during the developmental process; +tnd-1, +tnd-2, +cnrA, +cnrB, and +cnrD. The phenotypic expression of +tnd-1 began about 18 hr after nuclear determination and of +cnrB at about 24 hr at 27°C. However, phenotypic expression of the three other genes began about 40 hr after determination. It can be concluded that some of the genes of macronuclear anlagen begin to be expressed at or before the first measurable increase in the amount of DNA of the anlagen.

Developmentally Controlled Rearrangement of Surface Protein Genes in Paramecium tetraurelia1

ABSTRACT Early research on Paramecium genetics highlighted the role of the cytoplasm on inheritance. Today this tradition continues as recent investigations of macronuclear development in Paramecium have revealed unusual cytoplasmic effects that are not easily explained within current paradigms. It is generally assumed that most programmed DNA rearrangements in ciliates are regulated by cis acting signals encoded within the germline (micronuclear) DNA, but there are increasing examples in which the old macronucleus acts through the cytoplasm (in trans) to affect the loss and rearrangement of DNA in the developing macronucleus. The remarkable specificity of this effect has forced a reevaluation of the standard view of macronuclear determination in Paramecium. This review summarizes our knowledge of the effect of the old macronucleus on the developmentally controlled rearrangements of the P. tetraurelia, stock 51A and B variable surface protein genes.

Nuclear Dimorphism and Function

Ciliates have two kinds of nuclei, macronuclei and micronuclei, in a common cytoplasm. They differ both in structure and in function. The micronucleus is an ordinary diploid and plays the role of a germinal nucleus which undergoes meiosis and gives rise to gametic nuclei which form the zygotic nucleus (synkaryon) at fertilization. After fertilization the synkaryon gives rise to new micro-and macronuclei. Both micronuclei and macronuclei have typical nuclear envelopes, however, the chromatin of the micronucleus is densely packed and nucleoli are absent. The macronucleus is transcriptionally very active and is considered to control cytoplasmic and nuclear events. Does the micronucleus act only as a puppet of the macronucleus throughout the life cycle? The somatic function of the micronucleus has been debated for a long time. Recent work on Paramecium shows that the micronucleus plays an important role in stomatogenesis during both sexual and asexual phases of the life cycle. Nuclear dualism also provides unique and interesting problems for the study of nuclear function and differentiation.

Selection of the germinal micronucleus in Paramecium caudatum: nuclear division and nuclear death.

ABSTRACT. Each cell of Paramecium caudatum has a germinal micronucleus. When a bi‐micronucleate state was created artificially by micronuclear transplantation, both micronuclei divided for at least 2 cell cycles after nuclear transplantation. However, this bi‐micronucleate state was unstable and reduced to a uni‐micronucleate state after several fissions. Although the number of micronuclei was usually 1 during the vegetative phase, 4 presumptive micronuclei differentiated after conjugation. At the first post‐conjugational fission, only 1 of the 4 micronuclei divided, indicating that there is tight regulation of micronuclear number in exconjugants. Micronuclei that did not divide at the first post‐conjugational fission may persist through the first and second post‐conjugational cell cycles. The decision to divide appears to be separate from the decision to degenerate, as evidenced by division of a remaining micronucleus upon removal of the dividing micronucleus at the first division. Degeneration of micronuclei in exconjugants differs from that of haploid nuclei after meiosis. Nutritional state affected micronuclear degeneration. Under well‐fed conditions, the micronuclei destined to degenerate lost the ability to divide earlier than after starvation treatment, suggesting that micronuclear degeneration is an “apoptotic” phenomenon, probably under the control of the new macronuclei (macronuclear anlagen).

Microtubules mediate germ-nuclear behavior after meiosis in conjugation of Paramecium caudatum

Abstract Microtubule dynamics in Paramecium caudatum were investigated with an anti-α-tubulin antibody and a microinjection technique to determine the function of microtubules on micronuclear behavior during conjugation. After meiosis, all four haploid micronuclei were connected by microtubular filaments to the paroral region and moved close to this region. This nuclear movement was micronucleus-specific, because some small macronuclear fragments transplanted from exconjugants never moved to the region. Only one of the four germ nuclei moved into the paroral cone and was covered by microtubule assembly (the so-called first assembly of microtubules, AM-I). This nucleus survived there, while the other three not in this region degenerated. The movement of germ nucleus was inhibited by the injection of the anti-α-tubulin antibody. The surviving germ nucleus divided once and produced a migratory pronucleus and a stationary pronucleus. Prior to the reciprocal exchange of the migratory nuclei, microtubules assembled around the migratory pronuclei again (the so-called second assembly of microtubules, AM-II). Then, the migratory pronucleus moved into the partner cell and fused with the stationary pronucleus. Thus, microtubules appear to be indispensable for nuclear behavior: they enable migration of postmeiotic nuclei to the paroral region and they permit the survival of the nucleus at the paroral cone.

Repetitive Micronuclear Divisions in the Absence of the Macronucleus During Conjugation of Paramecium caudatum

ABSTRACT. The germinal micronucleus divides six times during conjugation of Paramecium caudatum: this includes two meiotic divisions and one mitosis of haploid nuclei during mating, and three mitoses of a fertilization nucleus (synkaryon). Microsurgical removal of the macronucleus showed that micronuclei were able to divide repeatedly in the absence of the macronucleus, after metaphase of meiosis I of the micronucleus and also after synkaryon formation. When the macronucleus was removed after the first division of synkaryon, in an extreme case the synkaryon divided five times and produced 32 nuclei, compared to three divisions and eight nuclei produced in the presence of the macronucleus. Treatment with actinomycin D (100 μ/ml) inhibited the morphological changes of the macronucleus during conjugation and induced a multimicronucleate state in exconjugants. However, in other cells, it induced production of a few giant micronuclei. We conclude that the micronucleus is able to undergo repeated divisions at any stage of conjugation in the absence of the macronucleus once the factor(s) for induction of the micronuclear division has been produced by the macronucleus. The macronucleus may also produce a regulatory factor required to stop micronucler division.

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.

Nuclear Behavior and Differentiation in Paramecium caudatum, Analyzed by Immunofluorescence with Anti-tubulin Antibody

Abstract To study the dynamics of the microtubules throughout the sexual cycle of Paramecium caudatum, we employed a monoclonal antibody (mAb) N356, that specifically recognized the 50-57 kDa axonemal proteins, equivalent to the tubulins of P. caudatum. The mAb decoration was observed on the micronuclei, as well as structures that consisted of microtubules not only in P. caudatum but also in other species P. bursaria, P. tetraurelia and P. trichium. While the micronucleus was consistently decorated with mAb, the macronucleus was decorated only during morphological changes, that is, at binary fission of the vegetative phase and at skein-formation during conjugation. During conjugation, the oral apparatus, with its associated microtubular structures, disappeared at meiosis I then reappeared at the end of the third division of the synkaryon. The current study led to several major findings on the behavior of the germinal micronucleus during conjugation: 1) the microtubules seemed to connect some of the haploid nuclei to the paroral region at the end of meiosis II; 2) mAb decoration disappeared from degenerating haploid nuclei, while it remained in the surviving nucleus; 3) mAb decoration also disappeared from macronuclear anlagen after the initiation of nuclear differentiation, while it remained in the other four post-zygotic micronuclei. The mAb decoration pattern could be used as an indicator of the fate of a micronucleus and suggests that both the intra-micronuclear and the cytoplasmic microtubules might have an important role in determining of the fate of micronuclei by translocating them to specific areas of the cytoplasm.

Characterization of the spasmin 1 gene in Zoothamnium arbuscula strain Kawagoe (protozoa, ciliophora) and its relation to other spasmins and centrins

Zoothamnium arbuscula strain Kawagoe is a giant sessile peritrich ciliated protozoa that possesses a contractile organelle called a spasmoneme. We report here on the molecular characterization and provide an opportunity to discuss the evolutionary relationships of the Z. arbuscula spasmin; spasmins belong to the calmodulin superfamily and are the major components of spasmoneme filaments. We analysed and obtained the whole sequence of the spasmin 1 gene and a partial sequence of the spasmin 2 gene. It is surprising that the sequence of spasmin 1 does not contain introns and encodes an open reading frame of 531 bp. It predicts a product of 177 amino acids with a calculated molecular mass of 19659 Da and a pI of 4.68. The amino acid sequence has two putative calcium-binding domains. One of them is a functional domain, as defined by the EF-hand consensus. The varieties of spasmins were revealed by comparison with amino acid components and molecular relationships of spasmin 1 protein and other spasmins. A comparison of the amino acid sequence between the Z. arbuscula spasmin and known centrins indicates that spasmins have a one residue deletion in the EF-hand domain-2 and four residue insertions in domain-4, as does the Vorticella spasmin. However, there are large variations in the amino acid sequence at domain-4 within spasmin 1, spasmin 2 and the Vorticella spasmin.