Abstract
DNA replication is a crucial process for species survival, nevertheless it is not clear which factors define origin selection in multicellular eukaryotes. Developmental gene amplification systems, such as the one described during ovarian follicles development in Drosophila melanogaster, are useful tools for studying of DNA replication process in these organisms. We previously described that the well characterized third chromosome amplified domain of D. melanogater displays three intrinsically bent DNA sites: b1, localized at an amplification control element (ACE3), b2 and b3, both localized at the preferential origin ori-β. This proposal aimed to construct a Drosophila transformation vector, which contains a short deletion at the ACE3, in order to reduce the intrinsically bent DNA site b1, and analyze the functional role of this site in the gene amplification process. Through a series of cloning steps, we obtained a Big Parent vector derivative, containing a deletion at the positions 176-180 bp, inside the ACE3. The generation of a Drosophila transformation vector displays a reduced intrinsically bent DNA site in the third chromosome amplified domain, it will allow the analysis of the functional role of this curvature in developmental gene amplification, providing new insights on replication initiation in D. melanogaster and the function of intrinsically bent DNA sites.
Highlights
The efficiency of the DNA replication process is essential for species survival
The machinery required for the replication process is well-known, the factors determining the selection and where is localized the origins which will be fired in a genome segment are still poorly understood, and a limited number of DNA replication origins had been studied in detail
The first step in the strategy to generate a transforming vector taking this unstructured intrinsically bent DNA site b1, was to construct a primer pair complementary introducing a deletion of 5 bp
Summary
The efficiency of the DNA replication process is essential for species survival. the genome should be quickly and faithfully duplicated only once per cell division. In the yeast Saccharomyces cerevisiae, the sites of initiation of replication (Autonomosly Replicating Sequences, ARS) include a short 10 to 11 bp consensus sequence rich in A/T called ACS (ARS consensus sequence) [3]. From this organism, during late M phase and early G1 phase of cell cycle, was isolated the ORC (Origin Recognition Complex), responsible for recognizing the sites of initiation of replication and nuclear components of pre-replicative complex (pre-RC), that are necessary to indicate permission for the initiation of replication in S phase [4]. The study of the DNA replication process is advantageous in both systems due to the high number of copies of DNA segments generated and the fact that the enzymatic machinery used is the same as described for regions not amplified [9]-[11]
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