James D. Forney, PhD

Area of Expertise: Regulation of differentiation in protozoa

Eukaryotic cell differentiation is generally considered a property of metazoa (multicellular organisms), yet dramatic examples of regulated cellular changes also occur in protozoa. Our laboratory is currently investigating the formation of the somatic macronucleus during sexual reproduction in ciliated protozoa.

Ciliated protozoa contain two types of nuclei, micronuclei and macronuclei, which arise from a single diploid fertilization nucleus during sexual reproduction. During the formation of the macronucleus, the germline chromosomes are amplified, fragmented and specific DNA sequences are eliminated. In Paramecium tetraurelia the coding regions of many micronuclear genes are interrupted by internal eliminated sequences (IES) that must be removed precisely to form the open reading frames in the macronuclear genome. These IESs range in size from 26 bp to over 1 kb and are always flanked by the dinucleotide 5'-TdA-3'. The isolation of mutant cell lines that are unable to remove a specific IES revealed nucleotides located within the first 6 base pairs of the eliminated sequence which are important for IES excision. Interestingly, this region contains a consensus sequence that has similarity to the termini of the mariner/Tc1 family of transposable elements. The possible relationship between this wide spread family of transposons and Paramecium IESs will require detailed knowledge of the cis and trans acting elements necessary for excision. An in vivo assay for IES excision is being used to dissect the cis acting sequences required for precise and efficient removal of these short DNA elements. With our collaborators we have shown that ligase IV and Xrcc4, proteins in the Non-Homologous End Joining (NHEJ) pathway, are required for elimination of IES in Paramecium. We also identified a novel ciliate specific protein (DIE5) that is required for IES excision in both Paramecium and the related ciliate Tetrahymena thermophila. Our focus is now turned to the role of SUMOylation in regulating critical events during formation of the macronuclear genome in Tetrahymena.