MS2 of Paramecium tetraurelia is the gene that we have cloned as one expressed at much higher levels in a short-lived mutant than in its parental wild-type stock and have characterized its expression to be enhanced with increased clonal age of the wild-type stock. This gene has been found to be preferentially expressed also in paramecia undergoing autogamy which is one of the termination modes of the Paramecium clonal life span. Here we investigated the transcriptional mechanism for the MS2 expression in light of its possible causal relationship to the Paramecium clonal life span. DNA-protein binding analyses of the upstream of the MS2 gene identified a stretch of DNA sequence that interacted specifically with macronuclear proteins prepared from the MS2- expressing mutant. The DNA sequence was mapped to the 33 bp between — 335 and — 303, counting from the initiation position of MS2 transcription. A protein, 15,000 in molecular mass, with a binding ability for this DNA element was purified to homogeneity from the macronuclear proteins by a chromatography using the specific DNA-protein interaction. In vitro transcriptional analyses revealed that both the purified protein and its target DNA domain are essential for increased synthesis of the MS2 transcript. These results showed that the DNA-protein interaction is required for induction of the MS2 expression.
In many kinds of protozoans, cyst formation (encystment) and excystment which involves structural transformation processes constitute the drastic process of cell reconstruction mediated by signaling chains including gene expression that is triggered by environmental signals. In order to establish a model system for understanding the molecular mechanisms of encystment and excystment, we aimed to elucidate the environmental factors that activate the signal transduction chain leading to encystment and excystment of Colpoda sp. The present study showed that cyst formation of Colpoda was repressed by bacteria, while accelerated by saline solution. The excystment of the cells was induced by certain components contained in dried cereal leaves.
The cAMP-dependent phosphorylation of the 29-kDa and 65-kDa axonemal proteins is closely correlated with the control mechanism of ciliary beat direction. Localization of the 65-kDa protein is still unknown whereas the 29-kDa protein has been identified as a light chain of the outer-arm 22S dynein. The localization of the 65-kDa phosphoprotein was determined by two-dimensional electrophoresis using a native gel in the first run. In the native gel, the 65-kDa phosphoprotein co-migrated with dynein heavy chains. This suggests that the 65-kDa phosphoprotein plays an important role in cAMP-dependent ciliary control as an intermediate chain of a dynein complex.
Previous studies on cell contraction in the ciliate Spirostomum have suggested that action may not be present, in spite of its ubiquity in other ciliates including Paramecium and Tetrahymena. In this study, different anti-actin antibodies were used to re-examine whether actin occurs in Spirostomum. Western blotting analysis with these anti-actin antibodies showed that actin-like proteins may exist in Spirostomum. Intracellular localization of actin-like proteins was examined by immunofluorescene microscopy with the same antibodies. Fluorescent signals were seen at the cleavage furrow, around the macronucleus, and at the ciliary bases including those of the membranelle. These results suggest that actin may exist in Spirostomum, but it is different from skeletal muscle actin and Tetrahymena actin in antigenic characteristics.