Transplantation of synkaryon in Paramecium caudatum. Analysis of its competence as germ nucleus

To establish whether the synkaryon of Paramecium can perform the function as a germ nucleus without a special process of differentiation into germ nucleus, synkaryons were transplanted into amicronucleate vegetative cells. The transplanted synkaryons underwent not only mitosis but also meiosis and the subsequent nuclear changes in the same way as the normal germ nucleus. The result suggests that no ‘determination’ of germ nucleus is necessary for the synkaryon to function as a germ nucleus. In Paramecium, the germ line is continuous through generations.     

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.     

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.     

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.     

DNA-membrane association during the mitotic cycle of Physarum polycephalum

Association of DNA with the nuclear membrane was studied in the naturally synchronous slime mold Physarum polycephalum. Sucrose gradient analyses showed that DNA is bound to the membrane during all stages of interphase. The association is labile, but can be stabilized by spermine, which presumably acts on the membrane. Experiments with pulse-labeled DNA suggest that replication does not occur at a membranal site.     

Gene conversion at different points in the mitotic cycle of Saccharomyces cerevisiae

Summary In the Saccharomyces cerevisiae mitotic cycle, the timing of radiation-induced gene conversion has been studied using thermosensitive cell division cycle mutants. The cells were found to perform conversion at different G1 or post-replication steps. A lower yield in induction is found during the G2 phase and is explained by the competition for recombinational repair between sister chromatids and homologous chromosomes. The results are discussed in relation to repair.     

Growth factor-deprived BALB/c 3T3 murine fibroblasts can enter the S phase after induction of c-myc gene expression.

Induction of quiescent BALB/c 3T3 murine fibroblasts by platelet-derived growth factor (PDGF) or fibroblast growth factor (FGFs) is accompanied by induction of c-myc gene expression. To study the role of c-myc in cell growth, we transfected BALB/c 3T3 cells with a plasmid construct containing a glucocorticoid-inducible c-myc gene. When these transfected cells were growth arrested in PDGF-FGF-freedefined medium, glucocorticoid treatment induced S-phase DNA synthesis. This induction of DNA synthesis was inefficient, and cell proliferation was not evident, suggesting that growth factors act through stimulation of c-myc expression together with other intracellular events.     

Arrest of the mitotic cell cycle and of meiosis in Saccharomyces cerevisiae by MMS

Summary Methyl methane sulphonate (MMS) was found to arrest mitotic cells at a specific stage in the cell cycle. Reciprocal double shift experiments involving MMS and temperature shifts in several temperature-sensitive cell-cycle (cdc) mutants have located the MMS-sensitive stage after the cdc7 and cdc8 temperature-sensitive stages and before the cdc13, cdc5 and cdc14 stages. An interdependent relationship was found between the arrests caused by MMS, cdc40 and hydroxyurea. Marked increases in mitotic recombination were induced by MMS, both in diploid and haploid strains. Meiosis is arrested by MMS at a very early stage, before DNA replication.     

The human cytomegalovirus IE86 protein can block cell cycle progression after inducing transition into the S phase of permissive cells

Human cytomegalovirus (HCMV) infection of permissive cells has been reported to induce a cell cycle halt. One or more viral proteins may be involved in halting progression at different stages of the cell cycle. We investigated how HCMV infection, and specifically IE86 protein expression, affects the cell cycles of permissive and nonpermissive cells. We used a recombinant virus that expresses the green fluorescent protein (GFP) to determine the effects of HCMV on the cell cycle of permissive cells. Fluorescence by GFP allowed us to select for only productively infected cells. Replication-defective adenovirus vectors expressing the IE72 or IE86 protein were also used to efficiently transduce 95% or more of the cells. The adenovirus-expressed IE86 protein was determined to be functional by demonstrating negative autoregulation of the major immediate-early promoter and activation of an early viral promoter in the context of the viral genome. To eliminate adenovirus protein effects, plasmids expressing GFP for fluorescent selection of only transfected cells and wild-type IE86 protein or a mutant IE86 protein were tested in permissive and nonpermissive cells. HCMV infection induced the entry of U373 cells into the S phase. All permissive cells infected with HCMV were blocked in cell cycle progression and could not divide. After either transduction or transfection and IE86 protein expression, the number of all permissive or nonpermissive cell types in the S phase increased significantly, but the cells could no longer divide. The IE72 protein did not have a significant effect on the S phase. Since IE86 protein inhibits cell cycle progression, the IE2 gene in a human fibroblast IE86 protein-expressing cell line was sequenced. The IE86 protein in these retrovirus-transduced cells has mutations in a critical region of the viral protein. The locations of the mutations and the function of the IE86 protein in controlling cell cycle progression are discussed.     

The 3' Untranslated Region of Messages in the Rumen Protozoan Entodinium caudatum

The 3' untranslated regions of a number of cDNAs from the rumen protozoal species Entodinium caudatum were studied with a view to characterising their preference for stop codons, general length, nucleotide composition and polyadenylation signals. Unlike a number of ciliates, Entodinium caudatum uses UAA as a stop codon, rather than as a codon for glutamine. In addition, the 3' untranslated region of the message is generally less than 100 nucleotides in length, extremely A+T rich, and does not appear to utilise any of the conventional polyadenylation signals described in other organisms.     

“Candidatus Paraholospora nucleivisitans”, an intracellular bacterium in Paramecium sexaurelia shuttles between the cytoplasm and the nucleus of its host

An intracellular bacterium was discovered in two isolates of Paramecium sexaurelia from an aquarium with tropical fish in Münster (Germany) and from a pond in the Wilhelma zoological–botanical garden, Stuttgart (Germany). The bacteria were regularly observed in the cytoplasm of the host, but on some occasions they were found in the macronucleus of the host cell. In these cases, only a few, if any, bacteria were observed remaining in the cytoplasm. The bacterium was not infectious to P. sexaurelia or other species of Paramecium and appeared to be an obligate intracellular bacterium, while bacteria-free host cells were completely viable. The fluorescence in situ hybridisation (FISH) and comparative 16SrDNA sequence analyses showed that the bacterium belonged to a new genus, and was most closely, yet quite distantly, related to Holospora obtusa. In spite of this relationship, the new bacteria differed from Holospora by at least two biological features. Whereas all Holospora species reside exclusively in the nuclei of various species of Paramecium and show a life cycle with a morphologically distinct infectious form, for the new bacterium no infectious form and no life cycle have been observed. For the new bacterium, the name Candidatus Paraholospora nucleivisitans is suggested. The host P. sexaurelia is usually known from tropical and subtropical areas and is not a species typically found in Germany and central Europe. Possibly, it had been taken to Germany with fish or plants from tropical or subtropical waters. Candidatus Paraholospora nucleivisitans may therefore be regarded as an intracellular neobacterium for Germany.     

Loss of heterozygosity in yeast can occur by ultraviolet irradiation during the S phase of the cell cycle

A CAN1/can1Δ heterozygous allele that determines loss of heterozygosity (LOH) was used to study recombination in Saccharomyces cerevisiae cells exposed to ultraviolet (UV) light at different points in the cell cycle. With this allele, recombination events can be detected as canavanine-resistant mutations after exposure of cells to UV radiation, since a significant fraction of LOH events appear to arise from recombination between homologous chromosomes. The radiation caused a higher level of LOH in cells that were in the S phase of the cell cycle relative to either cells at other points in the cell cycle or unsynchronized cells. In contrast, the inactivation of nucleotide excision repair abolished the cell cycle-specific induction by UV of LOH. We hypothesize that DNA lesions, if not repaired, were converted into double-strand breaks during stalled replication and these breaks could be repaired through recombination using a non-sister chromatid and probably also the sister chromatid. We argue that LOH may be an outcome used by yeast cells to recover from stalled replication at a lesion.     

A gene function required for cell cycle progression during the G1 portion of the cell cycle and for maintenance of macronuclear DNA synthesis in Paramecium tetraurelia

The ccl mutation in Paramecium tetraurelia reversibly and rapidly blocks cell cycle progression and DNA synthesis at the restrictive temperature. Progression through the cell cycle is blocked during both the G1 and S portions of the cell cycle, while at the restrictive temperature there is neither residual cell cycle progression nor induction of excess delay of subsequent cell cycle events. DNA synthesis activity is reduced to 50% of the normal level in about 5 min and is completely blocked at 30 min after a shift to restrictive temperature. On return to permissive conditions, DNA synthesis is reactivated with similar kinetics.