Gene amplification in Drosophila ovarian follicle cells as a developmental strategy and model for metazoan DNA replication

The process of gene amplification in Drosophila ovaries provides a means of increasing the amount of template for transcription, thus increasing the amount of protein that can be made over a short developmental period. At a specific developmental point (egg chamber stage l0B-13), several clusters of genes encoding the eggshell (chorion) proteins in the follicle cells of each egg chamber are overreplicated 20 or 60 fold (for the X chromosome and third chromosome amplicons, respectively). Gene amplification is accomplished using the normal eukaryotic DNA replication machinery and a bidirectional DNA replication mechanism, and as such, is a powerful system for the study of metazoan DNA replication. Furthermore, the nature of the ovaries, with egg chambers of various ages arrayed in the order they were created, coupled with the use of cell biology, allows for the visualization of gene amplification at multiple timepoints in a single sample. We employed confocal and deconvolution microscopy to visualize the replication proteins ORC2, DUP/Cdtl, PCNA, and MCM2-7, as well as the nucleotide analog BrdU, at sites of gene amplification. These studies revealed that the BrdU staining pattern resolves from a focus of incorporation at the third chromosome locus in egg chamber stage lOB, to a coffee-bean structure in stage 11 egg chambers, to a double-bar structure in egg chamber stages 12 and 13. When coupled with quantitative real-time PCR calculations of copy number at the third chorion cluster during egg chamber stages lOB-13, these studies demonstrated that amplicon origin firing ends by stage 11 and that only the existing replication forks move out during stages 12 and 13 to produce the double bar BrdU pattern.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Biology.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2004

Author dc:creator

• Claycomb, Julie Michelle, 1977-

Advisor dc:contributor.advisor

• Terry L. Orr-Weaver.

• Biology.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/31112

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/31112