Developmental, or programmed, DNA rearrangement events have been observed in a variety of organisms. In some instances, DNA rearrangement is used as a means of constructing protein coding regions, such as in the development of the vertebrae immune system, and can thus be viewed as a means of developmentally regulating gene expression. a number of pathogenic microorganisms also employ DNA rearrangement to alter their expression pattern of surface proteins as a means of evading the immune system. The molecular mechanisms involved in DNA rearrangement are poorly understood in eukaryotic organisms. To learn more about DNA rearrangement events, we propose to study the extensive genome reorganization process that occurs in the hypotrichous ciliated protozoan Euplotes crassus. During its life cycle, this organism transforms a copy of its chromosomal micronucleus into a macronucleus that contains only short, linear, gene-sized DNA molecules. The process of macronuclear development involves steps of chromosome fragmentation, DNA elimination, and DNA amplification. In addition, there are numerous DNA breakage and joining, or splicing, events that are associated with DNA elimination. Initial studies will examine the nature of the DNA breakage and joining events in greater detail so as to better understand their molecular mechanisms. This will include determining if the excision process is precise, characterizing the eliminated DNA, and determining the nature of the DNA breakage event involved in the excision process. Other studies are aimed at detecting proteins that interact with excised DNA sequences and determining the particular DNA sequences with which these proteins interact. This will be accomplished using a combination of gel mobility shift assays, in vivo DNA footprinting, and in vitro DNA footprinting. As an additional approach to identifying proteins involved in DNA rearrangement, a panel of monoclonal antibodies directed against nuclear proteins that are expressed during periods of DNA excision will be generated. These studies will provide new information on the cis-acting DNA sequences and trans-acting factors that direct the specific DNA breakage and joining events. In addition, these studies will lay the groundwork for future studies aimed at the isolation and characterization of the proteins that mediate the rearrangement process.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033277-09
Application #
2176942
Study Section
Genetics Study Section (GEN)
Project Start
1984-04-01
Project End
1994-12-31
Budget Start
1993-07-01
Budget End
1994-12-31
Support Year
9
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Connecticut
Department
Biochemistry
Type
Schools of Dentistry
DUNS #
City
Farmington
State
CT
Country
United States
Zip Code
06030
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Klobutcher, L A (1995) Developmentally excised DNA sequences in Euplotes crassus capable of forming G quartets. Proc Natl Acad Sci U S A 92:1979-83
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
Klobutcher, L A; Turner, L R; LaPlante, J (1993) Circular forms of developmentally excised DNA in Euplotes crassus have a heteroduplex junction. Genes Dev 7:84-94
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
Tausta, S L; Turner, L R; Buckley, L K et al. (1991) High fidelity developmental excision of Tec1 transposons and internal eliminated sequences in Euplotes crassus. Nucleic Acids Res 19:3229-36
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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