Title

The characterization of proteins expressed during macronuclear development in the ciliated protozoan Euplotes crassus

Date of Completion

January 1998

Keywords

Biology, Molecular|Biology, Genetics|Biology, Microbiology

Degree

Ph.D.

Abstract

The micronuclear-derived DNA in the developing macronucleus of the ciliated protozoan Euplotes crassus undergoes a massive reorganization during the 100 hour period that follows sexual mating. Rearrangement events during the polytene chromosome stage of development include the excision of thousands of short, unique-sequence regions of DNA called internal eliminated sequences (IESs) as well as the repetitive-sequence Tec transposon IESs. Conserved regions have been identified in the Tec transposon termini that may function in their excision. The first question addressed in this thesis is whether similar sequences exist within the short IESs. The results of a statistical analysis indicated that conserved sequences that may be required for excision are present in both the terminal and subterminal regions of the short IESs. The possible evolutionary relationship between the short IESs and Tec transposon IESs is discussed.^ The second question addressed was whether two recently isolated, developmentally-expressed genes may play a role in DNA excision. Full-length cDNA clones of the conZA7 and conZA8 genes were isolated and sequenced and data bases searched for homologs to their predicted gene products. The conZA7 gene is predicted to encode a basic protein of 193 amino acids. conZA7 also contains an acidic patch in its C-terminal region that suggests that this protein might function in transcriptional activation. No significant sequence similarities to any protein currently in the data bases have been found for conZA7.^ The conZA8 gene is predicted to encode an acidic protein of 468 amino acids. Antibodies raised against a recombinant form of conZA8 as well as against an internal peptide of the predicted protein were used in Western blotting and immunofluorescence analyses that indicated that conZA8 is expressed during the polytene chromosome stage of development and is localized to the developing macronucleus. To date, no significant sequence similarities to any protein currently in the data bases have been found for conZA8. However, the abundance of proline residues in its C-terminal region suggests that conZA8 may be involved in homotypic or heterotypic protein-protein interactions. Possible roles for conZA8 during the polytene stage of development are discussed. ^