Although a number of gene and genome rearrangement processes have been defined in eukaryotic organisms, the mechanisms that mediate these events are poorly understood. We plan to study the extensive genome reorganization process that occurs in hypotrichous ciliated protozoa, such as Oxytricha nova and Stylonychia mytilus, in order to extend our knowledge of eukaryotic rearrangement processes as well as to define the types of DNA sequences and enzymes that mediate such events. In these organisms a copy of the chromosomal micronuclear genome is transformed into a macronucleus containing small gene-size DNA molecules. This genome rearrangement process occurs at a defined time in the organism's life cycle and involves steps of DNA sequence elimination, sequence addition, and sequence rearrangement. We plan to study this genome rearrangement process by constructing recombinant DNA clones of macronuclear DNA molecules and the regions of the micronuclear chromosome from which they are derived. These materials will be characterized in order to define DNA sequences that are retained and eliminated during development. We will also clone and characterize intermediate DNA forms generated during the reorganization process in order to determine the sequence of events or steps involved in producing gene-size macronuclear DNA molecules from micronuclear chromosomes. Finally, we will develop two systems that will allow us to study the particular DNA sequences and enzymes mediating genome reorganization. In the first, we will directly microinject cloned micronuclear DNA sequences into developing macronuclei and follow their fate in order to precisely define sequences involved in rearrangement. Our second approach will be to develop in vitro assays for enzymes involved in rearrangement to ultimately purify and characterize their activities.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM033277-03
Application #
3282756
Study Section
Genetics Study Section (GEN)
Project Start
1984-04-01
Project End
1988-02-29
Budget Start
1986-04-01
Budget End
1988-02-29
Support Year
3
Fiscal Year
1986
Total Cost
Indirect Cost
Name
University of Connecticut
Department
Type
School of Medicine & Dentistry
DUNS #
City
Farmington
State
CT
Country
United States
Zip Code
06030
Ling, Z; Ghosh, S; Jacobs, M E et al. (1997) Conjugation-specific genes in the ciliate Euplotes crassus: gene expression from the old macronucleus. J Eukaryot Microbiol 44:1-11
Hale, C A; Jacobs, M E; Estes, H G et al. (1996) Micronuclear and macronuclear sequences of a Euplotes crassus gene encoding a putative nuclear protein kinase. J Eukaryot Microbiol 43:389-92
Ghosh, S (1996) A novel ligation mediated-PCR based strategy for construction of subtraction libraries from limiting amounts of mRNA. Nucleic Acids Res 24:795-6
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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