The current application emphasizes genetic approaches to the study of hsr-omega. It has three specific aims.
The first aim i s to isolate and characterize a deletion mutation that affects only hsr-omega. P element insertions within 1.5 kb of the hsr-omega transcription initiation site will be mobilized to obtain imprecise excisions of these insertions. Chromosomes from which these elements have been excised will be tested for a phenotypic effect in combination with a deletion of hsr-omega and several adjacent genes. The excision chromosomes will also be analyzed by Southern blotting to determine if they carry deletions of the hsr-omega gene. If a knock-out deletion of the gene is obtained, Dr. Pardue will proceed to ascertain its effect in homozygous cell clones generated by the FLP recombination system. She will also try to rescue whatever phenotypic effect such a deletion has by combining it with an hsr-omega transgene.
The second aim i s to investigate hsr-omega function by over-expressing and miss-expressing hsr-omega RNA. The experiments will utilize complete and modified hsr-omega sequences fused to the GMR promoter, a control element that activates transcription in the developing eye. Dr. Pardue will examine transgenic flies carrying these fusion constructs for perturbation in eye development. Prior to these experiments, Dr. Pardue will evaluate each of the constructs for expression in cultured insect cells.
The third aim i s to investigate the relevance of transcriptional silencing of hsr-omega in primary spermatocytes and the reactivation of hsr-omega in spermatids. To this end, Dr. Pardue will construct fusions of hsr-omega DNA and the b2-tubulin promoter, a control element that specifically directs gene expression in the male germ line, and insert these constructs into flies. She will then test the transgenic stocks for male fertility.
