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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033277-06
Application #
3282758
Study Section
Genetics Study Section (GEN)
Project Start
1984-04-01
Project End
1991-06-30
Budget Start
1989-12-01
Budget End
1991-06-30
Support Year
6
Fiscal Year
1990
Total Cost
Indirect Cost
Name
University of Connecticut
Department
Type
Schools of Dentistry
DUNS #
City
Farmington
State
CT
Country
United States
Zip Code
06030
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Ghosh, S; Jaraczewski, J W; Klobutcher, L A et al. (1994) Characterization of transcription initiation, translation initiation, and poly(A) addition sites in the gene-sized macronuclear DNA molecules of Euplotes. Nucleic Acids Res 22:214-21
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Baird, S E; Klobutcher, L A (1991) Differential DNA amplification and copy number control in the hypotrichous ciliate Euplotes crassus. J Protozool 38:136-40
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Klobutcher, L A; Turner, L R; Peralta, M E (1991) Sequence of a Euplotes crassus macronuclear DNA molecule encoding a protein with homology to a rat form-I phosphoinositide-specific phospholipase C. J Protozool 38:425-7
Tausta, S L; Klobutcher, L A (1990) Internal eliminated sequences are removed prior to chromosome fragmentation during development in Euplotes crassus. Nucleic Acids Res 18:845-53

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