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 the hypotrichous ciliated protozoan Euplotes crassus 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. The macronucleus contains small linear DNA molecules, each carrying a different gene. The formation of the macronuclear DNA molecules involves recognition of specific fragmentation sites and addition of telomeres, precise elimination of sequences internal to the retained macronuclear sequences and degradation of the majority of the micronuclear genome. This process can be studied in Euplotes crassus because large quantities of two different mating types can be grown which when mixed undergo synchronous mating, meiosis, and macronuclear development. We have identified a highly abundant (>10(4) copies per genome) repetitive element family (Tec-1) that frequently interrupts the macronuclear-destined sequences. The element excises during a discrete time period of macronuclear development and can be found as extrachromosomal circles. The abundance of this element and its excised form coupled with the precise timing of excision provide an ideal starting point for molecular analyses of the mechanisms behind this genomic reorganization process. Our goal is to determine how this process is controlled such that excision occurs in a precise manner at a specific stage of macronuclear development. Our experimental aims include: a) DNA sequence analysis of the element's inverted repeats in its integrated and excised forms; b) analysis of element gene expression; c) identification of element encoded gene products and their role in excision; d) identification of nonelement encoded proteins that are involved in excision; and e) examining the re- lationship of DNA replication and methylation to the timing of Tec-1 excision.
Showing the most recent 10 out of 13 publications
