The aim of this proposal is to create novel reagents for the Drosophila research community to facilitate mapping and identification of genes on the X chromosome and to facilitate manipulation of large autosomal genes. Mapping of genes on the autosomes has been greatly facilitated by genome-wide projects to generate molecularly defined deletions. Unfortunately, deletion mapping is not easy to apply to the X chromosome, as males carry only one copy and so are hemizygous. The alternative to deletion mapping is duplication mapping, i.e. one can use duplications of the X chromosome that are translocated onto the Y chromosome or an autosome to map essential genes on the X chromosome. If these duplications are molecularly defined, as is the case for the deletions, then one can map a gene quickly and precisely. The simplest way to create such a set of defined duplications would be to generate a collection of overlapping large transgenic fragments that cover the entire X chromosome and are inserted onto the Y chromosome or an autosome. Here, we propose to create 350 fly stocks that carry molecularly defined ~100 kb transgenic fragments inserted on the Y chromosome and that cover the entire euchromatic portion of the X chromos- ome. To achieve this goal, we have developed a set of seminal new tools by introducing recombineering technology into a set of vectors (the Pfacman] vectors) that allow transformation via phiC31-mediated integration. We show that genomic constructs in the 75-133 kb range can be engineered through recomb- ineering mediated gap-repair in bacteria, that these DNA fragments can be easily manipulated, and that they can be inserted into the genome efficiently. Hence, we propose to construct a BAG library with a P[acman] vector, sequence and map the ends of the cloned inserts, and create transgenic fly stocks. These reagents will greatly facilitate mapping and identification of genes on the X chromosome, one of the key priorities in the current Drosophila white paper. We also propose to identify or engineer 220 P[acman] BACs to create transgenic strains that carry large autosomal or heterochromatic genes. Mutations in large genes cannot be rescued by P element transgensis, but can easily be manipulated by recombineering and inserted efficiently into the genome through phiC31-mediated integration. All the vectors, the BAG library, and the transgenic stocks will be made available to the research community as soon as they are generated and tested. ? ? ?
| Ugur, Berrak; Bao, Huan; Stawarski, Michal et al. (2017) The Krebs Cycle Enzyme Isocitrate Dehydrogenase 3A Couples Mitochondrial Metabolism to Synaptic Transmission. Cell Rep 21:3794-3806 |
| Venken, Koen J T; Popodi, Ellen; Holtzman, Stacy L et al. (2010) A molecularly defined duplication set for the X chromosome of Drosophila melanogaster. Genetics 186:1111-25 |
| Venken, Koen J T; Carlson, Joseph W; Schulze, Karen L et al. (2009) Versatile P[acman] BAC libraries for transgenesis studies in Drosophila melanogaster. Nat Methods 6:431-4 |
