Dr. Golic has engineered an extremely powerful site- specific recombination system for Drosophila that exploits the FLP site- specific recombination system of the yeast 2-micron plasmid. With this system, recombination between two FRT DNA sites is induced in Drosophila following the heat-shock stimulated expression of a transgene carrying the yeast FLP recombinase gene driven by the hsp70 promoter. This system is currently being used by many laboratories in screens that rely on high frequency, non-deleterious mitotic recombination, but it has many other potential genetic and developmental applications, several of which Dr. Golic proposes here to continue exploring. There are two sections to this proposal. One section deals with the development of site- specific recombination technology itself, while the other applies that technology to address a biological question: why does the translocation of more than half of an X chromosome to any of the autosomes cause Drosophila males to be infertile? Three areas of site-specific recombination technology are covered in the first part of the proposal. First, Dr. Golic will attempt to improve the efficiency of targeted DNA integration by optimizing the following three steps that are involved: (1) the pairing of incoming DNA with the target site; (2) recombination between incoming DNA and the target; and (3) retention of the inserted DNA at the target. Targeted DNA integration is useful for many reasons, including the placement of engineered transgenes in a constant chromosomal environment. Representative examples of experimental questions addressed are: (a) To what extent does flanking homology affect pairing? (b) Can synthesis of FLP recombinase be induced very late in the germ line when the number of opportunities for independent events are greater? and (c) Can FLP recombinase and/or the FRT element itself be partially disabled in ways that would decrease problems caused by re-excision? Second, Dr. Golic will continue to explore the usefulness of the FLP system for efficiently generating specific chromosome rearrangements. Towards this end, attempts will be made to develop efficient methods for ascertaining the chromosomal orientation of integrated FRT elements, methods will be developed to overcome the problem of segregation of interchanged chromosomes, and the FRT vectors will be improved that, when integrated into the chromosome, generate the rearrangements. Third, Dr. Golic will attempt to combine the FRT system with a site- specific DNA cleavage system from yeast, the HO system involved in mating-type conversion, in order to develop efficient methods for achieving homologous integration of DNA introduced into the germline. Success in this area would allow the power of reverse genetics to be applied to Drosophila to its fullest extent, and would thereby help those working with this organism to catch up to yeast and mouse investigators in this important aspect of genetic technology. The second part of the proposal deals with question of why translocations of large portions of the X chromosome to the autosomes are incompatible with spermatogenesis. Four experimental approaches are presented. First, Dr. Golic will attempt to test the hypothesis that such sterility results from interference with precocious X chromosome inactivation. Using a beta2-tubulin driven FLPase source, he will determine whether translocations induced very late in spermatogenesis, but still prior to X inactivation, cause sterility. Second, he will systematically characterize freshly generated male-sterile T(1;A)'s to ascertain whether the magnitude of the disruption in spermatogenesis is strictly proportional to the amount of X chromosome material translocated, and to determine just what the phenotypic series of increasingly deleterious effects on fertility looks at a relatively high level of cytological resolution. Third, he will explore the autonomy, timing, and reversibility of T(1;A) male sterility. Lastly, he will attempt to isolate recessive single gene suppressors of T(1;A) male sterility.
