NEW METHODS FOR MANIPULATION OF THE DROSOPHILA GENOME

Florida State University, Tallahassee FL

Investigators

Abstract

DESCRIPTION (Adapted from the Investigator's Abstract): The immediate goal of the proposed research is to construct large, "extended" P elements containing large regions of the Drosophila genome between two "half-P" elements and to characterize these extended P elements in terms of transposase-mediated events. Half-P elements were identified in the 66DE region of the genome using a novel genetic screen and used to construct a prototypical extended P element of approximately 100 kb. The investigators have demonstrated that this P element, approximately twice the size of any previously constructed, is capable of transposition to new sites in the genome at easily detectable frequencies. The investigators now propose to create a larger set of extended P elements on the second chromosome using hermes-based transformation vectors to introduce and mobilize independent transgenes containing the 5' and 3' ends of the P element. The insertion sites and orientations of the half-P insertions will be determined by comparison of DNA sequences obtained from flanking DNA to available genomic sequences and used to identify a set of 20-30 potential partner half-P elements based on chromosomal location and orientation. Meiotic recombination will then be used to construct a representative set of approximately 20 extended P elements which will be mobilized, and the resulting transposition and excision/deletion events analyzed genetically by using complementation tests, and molecularly by long-range restriction mapping and in situ hybridization. This analysis will allow the investigators to assess the practicality of scaling up this procedure for the Drosophila genome and to determine how to best use extended P elements for functional analysis of the Drosophila genome. Two potentially valuable applications of this methodology would be the construction of matching sets of transpositional duplications deletions and the direct construction of complementation maps by genetic analysis of extended P element excision and transposition events.