The long-term aim is to learn the mechanisms and significance of somatic genome alterations in a system where readily studied, namely, ciliated protozoa. Developmental somatic genome alterations play a central role in the immune system, and unscheduled alterations are a major component in the etiology of cancer. In ciliated protozoa, the somatic nucleus or macronucleus (MAC) is replaced each sexual cycle with a new MAC derived from the germline micronuclei (MICs) of the conjugating parents. Development of the new MAC involves drastic remodeling of its genome, including precise excision of 10(4)-10(5) segments called Internal Eliminated Sequences (IESs) and formation of 10(4)-10(5) new telomeres. The new, edited MAC is responsible for transcription and the unprocessed germline is silent. Two major theses are to be tested. First, chromosome breakage by a single mechanism may underlie both IES excision and telomere formation. Various rearrangement sites of both types will be examined to test whether they share a common consensus sequence, and to learn the fates of the ends creates at breaksites, to test for instance, if the two ends created by a single break have independent fates. While some IES are known to form circles prior to elimination, the TBE1 transposon-like IES are predicted to form linear """"""""telomered"""""""" chromosomes. The form of excised TBE1s will be determined. Second, transposon-like elements, such as the TBE1 elements, have been discovered in the germlines of different ciliates, and in some cases have been shown to be IESs interrupting MAC expressed genes. Precisely excisable elements should enjoy a fitness advantage, because they can interrupt germline genes which are restored in the MAC. Several predictions will be tested. Are TBE1s actually transposons? Have other MIC-limited transposon-like elements evolved to be IESs? It is argued that all the 10(4)-10(5) IESs are highly degenerate transposon family members which have only retained those sequences necessary for their precise excision: are degenerate TBE1 elements also IESs? The precise excision machinery may be encoded by one or more transposon families. Evidence for transcription of TBE1 genes will be sought in developing MACs. A TBE1-encoded protein with zinc fingers and an apparent protein kinase domain will be studied in vitro to test for specific binding to TBE1 sequences and for phosphorylation of other TBE1-encoded proteins. A system will be developed to allow dissection of the mechanism of IES excision. Plasmid-cloned IES-bearing MIC DNA substrates will be exposed to machinery in developing MACs. Harvested plasmid DNA will be assayed for IES excision by sensitive methods, to allow detection of modest amounts of product and optimization of the system. The system will allow development of assays for other types of MAC rearrangement, and the development of MAC transformation systems to determine MAC DNA features necessary for replication, control of copy number, and control of gene expression.
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