Analysis of metazoan deoxyribonucleic acid replication initiation using Drosophila gene amplification as a model system

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, February 2011.Cataloged from PDF version of thesis.Includes bibliographical references.Gene amplification in Drosophila follicle cells is an excellent model to study origin specification and developmental regulation of DNA replication in vivo. We mapped all follicle cell amplicons using a comparative genomic hybridization strategy and identified two new amplicons. We determined the precise localization of the origin recognition complex (ORC) on a genome-wide level and observed that, at the start of synchronous amplification, ORC localizes to the six amplicons with levels corresponding to the magnitude of amplification. Additionally, we investigated amplification with respect to transcription and chromatin state. The levels and timing of gene expression in some amplicons suggest that gene amplification is not exclusively a developmental strategy to promote high expression levels. Follicle cell amplicons are enriched for acetylated H4, but this mark is not sufficient for ORC localization or amplification. In addition to genome-wide analyses, we characterized the two new amplicons and discovered unique properties that make both distinctive replication models. Strikingly, DAFC-22B shows strain-specificity in amplification, a property that is correlated with the ability to localize ORC. We identified sequence differences between closely related amplifying and non-amplifying strains and used P element mediated transformation to test sufficiency for ORC binding and amplification at this region. DAFC-34B contains two genes that are expressed in follicle cells. Vm34Ca is a structural component of the vitelline membrane but is expressed prior to the onset of gene amplification. CG16956 is expressed in amplification stages but only in a small subset of follicle cells. Like the previously characterized DAFC-62D, DAFC-34B displays origin firing at two separate stages of development. However, unlike DAFC-62D, amplification at the later stage is not transcription dependent. We mapped the DAFC-34B amplification origin to 1kb by nascent strand analysis and delineated the cis requirements for origin activity, finding that a 6 kb region, but not the 1 kb origin alone, is sufficient for amplification. We analyzed the developmental localization of ORC and the MCM complex, the replicative helicase. Intriguingly, the final round of origin activation at DAFC-34B occurs in the absence of detectable ORC, though MCMs are present, suggesting a novel initiation mechanism. Our analysis of follicle cell amplicons highlights the diversity of amplification origin control mechanisms within the same cell type, which may be representative of similar regulatory diversity during S phase DNA replication.by Jane Christina Kim.Ph.D