Genomic rearrangements are known to be associated with human diseases, particularly birth defects and cancer. By studying a controlled genomic rearrangement process such as that occurring in hypotrichous ciliated protozoa during the formation of a macronucleus, we can determine what DNA sequences and enzymes might be involved and how the process is controlled. Hypotrichous ciliated protozoa dramatically reorganize their DNA in the process of forming a transcriptionally active macronucleus from an inactive micronucleus. Typical eukaryotic chromosomes are found only in the micronucleus. The macronucleus contains """"""""minichromosomes"""""""": small linear DNA molecules, each carrying a different gene, each with telomeric repeat sequences at its ends, and ach organized in a typical eukaryotic chromatin structure. Formation of these minichromosomes can be easily studied in the organism Euplotes crassus because different mating types can be grown in large quantities and mixed, then synchronous mating, meiosis, and macronuclear development ensue. The developing macronuclei can be isolated at points throughout this reorganization process.
The aims of the proposed studies are to determine: (a) how macronuclear sequences are excised from the micronuclear chromosomes; (b) what sequences are recognized in the excision process; (c) how telomeric repeat sequences are added to the ends of macronuclear sequences and are later processed to their final size; and (d) whether polytenization of the micronuclear chromosomes plays a role in defining what sequences are eliminated or retained. Methods to be used are: (a) Southern blot analysis of developing macronuclear DNAs using cloned micronuclear sequences as probes; (b) cloning and DNA sequencing of intermediates and junctions of retained and eliminated DNA; (c) sizing of telomeric repeat sequences using S1 nuclease and DNA sequencing; (d) assaying telomere addition and trimming activities in extracts made from developing macronuclei.
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