Nobuyuki Haga

Expression of telomerase reverse transcriptase and telomere elongation during sexual maturation in Paramecium caudatum

Paramecium caudatum has a sexually immature period that lasts for about 60 fissions. To examine the possibility that telomere length is one of the determining factors of the duration of immaturity, we cloned the telomerase reverse transcriptase (TERT) gene from P. caudatum, and analyzed its expression levels at mRNA, telomerase activity, and telomere length during the course of clonal division. Paramecium TERT (Pc_TERT) cDNA encodes a basic protein of 107 kDa that harbors conserved RT motifs, T motif, CP motif, and N motif. Pc_TERT mRNA is expressed at very low levels only detectable by RT-PCR, but constitutively, during immature and mature periods, exhibiting abundant telomerase activity. No clear phase shift in Pc_TERT expression, telomerase activity, or telomere length was observed at the point of maturation in P. caudatum. Instead, the telomere elongates successively as cells divide in P. caudatum, although a close species, P. tetraurelia, was reported to keep the length constant. We discuss possible mechanisms for the expression of sexual activity associated with telomere length in P. caudatum.

Studies of Paramecium caudatum by means of scanning electron microscope and projection X-ray microscope.

Samples of Paramecium caudatum are observed by means of a scanning electron microscope (SEM) and a projection X-ray microscope (XRM) with computer tomography (CT) function. The samples are fixed with two kinds of fixatives, glutaraldehyde and osmium-tetra oxide acid. After the fixation and replacement procedure with t-buthyl alcohol, the samples followed by a freeze drying, well retain their structures. Surface structures, cilia and microfibrillar systems including infraciliary lattice structures, are clearly depicted by SEM observation. On the other hand, XRM images give quite different information, namely, in the case of osmium oxide fixation, the structures of internal organelles like the macronucleus placed in the central part of cell body and trichocysts located under the cell membrane of a whole body are visible. In the case of glutaraldehyde fixation, the surface structures and internal structures are both visible but their image contrast is fairly weak. In order to examine toxicological effect, Paramecium caudatum samples treated in the environmental condition containing nano-particles of Ag (17 nm across) and Co-ferrite (300 nm across) are observed with results of certain morphological differences, namely, inner vacuoles increase in number and in volume in Co-ferrite treated cells as compared with Ag treated ones. But then, cilia-less areas increase on the surface of the body of Ag treated cells. In the case of Co-ferrite treated cells, cilia-less areas are not clearly detected. Whether these morphological differences observed in Ag and Co-ferrite treated cells are caused by the differences of materials or particle sizes remain to be examined in future.

Identification of Two Nickel Ion-Induced Genes, NCI16 and PcGST1, in Paramecium caudatum

ABSTRACT Here, we describe the isolation of two nickel-induced genes in Paramecium caudatum, NCI16 and PcGST1, by subtractive hybridization. NCI16 encoded a predicted four-transmembrane domain protein (∼16 kDa) of unknown function, and PcGST1 encoded glutathione S-transferase (GST; ∼25 kDa) with GST and glutathione peroxidase (GPx) activities. Exposing cells to cobalt chloride also caused the moderate upregulation of NCI16 and PcGST1 mRNAs. Both nickel sulfate and cobalt chloride dose dependently induced NCI16 and PcGST1 mRNAs, but with different profiles. Nickel treatment caused a continuous increase in PcGST1 and NCI16 mRNA levels for up to 3 and 6 days, respectively, and a notable increase in H2O2 concentrations in P. caudatum. NCI16 expression was significantly enhanced by incubating cells with H2O2, implying that NCI16 induction in the presence of nickel ions is caused by reactive oxygen species (ROS). On the other hand, PcGST1 was highly induced by the antioxidant tert-butylhydroquinone (tBHQ) but not by H2O2, suggesting that different mechanisms mediate the induction of NCI16 and PcGST1. We introduced a luciferase reporter vector with an ∼0.42-kb putative PcGST1 promoter into cells and then exposed the transformants to nickel sulfate. This resulted in significant luciferase upregulation, indicating that the putative PcGST1 promoter contains a nickel-responsive element. Our nickel-inducible system also may be applicable to the efficient expression of proteins that are toxic to host cells or require temporal control.

Induction of mating-type change in a non-selfer mutant of Paramecium by microinjecting wild-type genomic DNA

Summary Mating-type change in the life cycle of Paramecium caudatum is based on intrinsic tem poral programming related to clonal aging. Approximately 80–120 fissions after conjugation, some of the E-type cells change their mating-type to O-type, resulting in an intraclonal mating reaction and in selfing-pair formation. We have found that a natural stock, C103, is a non-selfer mutant with the homozygote of a recessive allele. Microinjection of genomic DNA isolated from selfer strains induced a mating-type change in the non-selfer mutant, C103. These results were consistent with those of previous studies showing that a dominant modifier gene, Su(Mt), suppresses the stable expression of the Mt gene. The present report represents the first demonstration that microinjection of genomic DNA restores the genetic defect related to stable expression of mating type in Paramecium.