GENOME REORGANIZATION IN HYPOTRICHOUS CILIATED PROTOZOA

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Programmed genomic rearrangement events have been observed in a variety of prokaryotic and eukaryotic organisms. In many instances, DNA rearrangement is used to control gene expression, as in the development of the vertebrate immune system. In addition, some pathogenic microorganisms utilize DNA rearrangement as a means of regulating the expression of their surface proteins so as to evade the immune response. Extensive developmental genomic rearrangement events, involving the elimination of whole chromosomes or parts of chromosomes, have also been observed, but their function is unknown. The proposed studies will examine the genome reorganization events of macronuclear development in the hypotrichous ciliated protozoa Oxytricha nova and Euplotes crassus. During their life cycle, these organisms transform a copy of their chromosomal micronucleus into a macronucleus containing linear, gene-sized DNA molecules. The process of generating a macronucleus involves a number of types of rearrangement events including chromosome fragmentation, DNA elimination, nucleic acid splicing, and DNA sequence addition. The extensive nature of the rearrangement process, and the ability to control it in the laboratory, make this a favorable model system for studying DNA rearrangement in eukaryotes. Initial studies will make use of characterized recombinant clones of macronuclear DNA molecules and their micronuclear precursors to analyze the timing and order of the various rearrangement events during macronuclear development. These studies will be performed on E. crassus, which can be used to generate large numbers of cells synchronously proceeding through macronuclear development, thus providing a source of staged DNAs. Additional studies will define chromosomal sites which impart specificity to the rearrangement process. The method to be used involves microinjection of cloned DNA molecules into cells undergoing macronuclear development,followed by reisolation and analysis of the DNA to detect processing. Nuclear extracts will also be prepared from cells undergoing development and used to develop in vitro systems for either detecting enzymes catalyzing rearrangement events or proteins which interact with chromosomal rearrangement sites. These studies will provide new information on DNA sequences and proteins involved in DNA rearrangement events and extend our knowledge of the molecular mechanisms of genome reorganization.