Herstellung von Knock-out Mutanten in Paramecium tetraurelia am Beispiel des Exocytose-sensitiven Phosphoproteins PP63/Parafusin

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This aim of this proposal is to establish a reliable method for gene knock-outs in P. tetraurelia to elucidate the function of its enzymes or proteins in a given cellular process. Unlike mammalian cells, this single-celled organism contains two morphologically and functionally distinct types of nuclei: two diploid transcriptionally silent germ-line micronuclei and a highly amplified transcriptionally active macronucleus. Based on several observations at the time of sexual reproduction, when a new macronucleus is developed from the micronucleus-derived germline by genomic rearrangement and amplification, I have devised three strategies to create stable mutant cell lines deficient in the well-characterized exocytosis-sensitive phosphoprotein, PP63/parafusin (phosphoglucomutase), the function of which in exocytosis regulation is not known. The first strategy is to disrupt the two PP63/parafusin genes with their internal eliminated sequences (IES). This was aborted after my experiments showed that no IES is present in the germline coding and 5' flanking sequences. The second is to interrupt the germ-line sequences with a heterologous marker gene. For this, I have developed protocols for transformation en masse and established at least one suitable non-invasive and sensitive reporter. The third strategy is to disrupt the genes by homology-dependent macronuclear deletions. Complex as this project has been, an extension will allow me to bring it to a fruitful conclusion.