Screening of the Paramecium tetraurelia Megabase chromosome for autogamy-specific genes.

Jacek Nowak#*, Marek Juszczuk*, Robert Gromadka*, Maria Jerka-Dziadosz§, Marie-Hélène Mucchielli=, Olivier Arnaiz#, Nicolas Augier=, Thomas Tang=, Lawrence Aggerbeck#=, Jean Cohen#, Hervé Delacroix=, Christopher Herbert#, Linda Sperling#, Mireille Bétermier#+, Marek Zagulski*

* Institute of Biochemistry and Biophysics, PAS, Warsaw, Poland, + Laboratoire de Génétique Moléculaire, ENS, Paris, France, # Centre de Génétique Moléculaire, CNRS, Gif-sur-Yvette, France, = The Gif-Orsay DNA Microarray Platform, Gif-sur-Yvette, France § The Nencki Institute of Experimental Biology, PAS, Warsaw, Poland

The macronuclear genome of Paramecium has been sequenced recently by the Genoscope (Aury et al., 2006). As a pilot project for the design and hybridization of Paramecium DNA microarrays, we focused on the 463 genes carried by the first macronuclear chromosome that has been annotated manually, the Megabase chromosome (Zagulski et al., 2004). The DNA microarrays containing 439 PCR-generated probes covering the majority of Megabase chromosome-encoded genes were hybridized with fluorescently labelled cDNA isolated from Paramecium cells at different stages of the sexual cycle. Statistical analysis of the results grouped microarray probes into 20 clusters of similar patterns of expression. Afterwards we noticed that some clusters can be grouped together into larger clusters showing similar characteristic profiles of expression. We found four groups: with maximum expression at T0 (early autogamy), with maximum expression at T11 (late autogamy), repressed during autogamy and gradually induced during autogamy. The T0 peak, the T11 peak and the repressed cluster contained approximately 30 megabase probes each and 14 probes were assigned to the induced cluster To validate the biological significance of the microarray data, RNAs were extracted during an independent time course experiment performed with different Paramecium stock and Northern blot hybridization was conducted for selected genes. The T0 peak, the T11 peak and the repressed clusters were clearly validated by Northern blots. The fourth cluster, containing gradually induced genes, could not be validated experimentally for all genes. To get an insight into the fraction of induced genes from the T0 cluster that may play an essential role during autogamy, we performed a functional RNAi-based screening of the effect of inactivating their expression both during vegetative growth and, following starvation, during autogamy. The phenotypes examined were growth rate, cell shape and survival. For the genes which did not appear to be essential for vegetative growth, cells were starved in feeding medium to induce autogamy and survival of post-autogamous cells was monitored following transfer into standard rich medium. Out of the 19 genes tested in this small scale screen, 5 were essential during vegetative growth. Only two were found to be essential to produce viable sexual progeny.