In situ hybridization as a method to study the regulation of gene expression in Paramecium

A method for whole cell in situ hybridization in Paramecium was developed, which allowed us to visualize both the cytoplasmic mRNA and the transcripts in the macronucleus, and to estimate their respective variation throughout the cell cycle. Nonisotopic DNA probes, labeled with digoxigenin (DIG), were prepared by PCR from a central part of two genes of P. tetraurelia, respectively coding for a beta-tubulin and the 51A surface antigen (51A-sAg). The probes were detected with an FITC-conjugated anti-DIG antibody. While no fluorescence is observed in the micronuclei, mRNA are detected in the macronucleus, as discrete spots which disappear after RNase H treatment, in the cytoplasm where they are uniformly distributed, and in the post-autogamous fragments of the old macronucleus. The intensity of the cytoplasmic labeling observed with the two probes shows a clear and different cell cycle-dependent modulation. For the beta-tubulin mRNA, a peak is observed at the beginning of mitosis, while for the 51A-sAg, the peak occurs later, during cytokinesis. The macronuclear labeling, which likely reflects the transcription rate, seems roughly constant for the 51A-sAg. In contrast, for the beta-tubulin genes, the intensity of the macronuclear signal shows a peak that just precedes the cytoplasmic peak.