GENETIC STUDIES ON AN UNUSUAL RNA CODING GENE, HSR-OMEGA

目前的应用强调用遗传学的方法来研究 HSR-omega。它有三个具体目标。第一个目标是隔离和 描述一种仅影响HSR-omega的缺失突变。P元素 在HSR-omega转录起始点1.5kb内插入将 动员起来，以获得这些插入物的不精确切除。染色体 从其中去除这些元素的基因将进行表型测试 结合HSR-omega和几个相邻基因的缺失的影响 基因。切除的染色体还将进行Southern blotting分析 以确定它们是否携带HSR-omega基因的缺失。如果一次击倒 如果基因被删除，帕杜博士将着手确定其 对FLP重组系统产生的纯合细胞克隆的影响。 她还将试图挽救这种缺失所产生的任何表型影响 通过将其与HSR-omega转基因相结合。 第二个目的是研究HSR-omega的功能，通过过度表达和 缺失表达的HSR-omega RNA。实验将利用完整的和 修饰的HSR-omega序列与GMR启动子(控制元件)融合 这会激活发育中眼睛的转录。帕杜医生将检查 携带这些融合结构的转基因果蝇在眼睛中的扰动 发展。在这些实验之前，帕杜博士将评估每一个 在培养的昆虫细胞中表达的构建物。 第三个目的是研究转录沉默的相关性。 HSR-omega在初级精母细胞中的表达及HSR-omega的再激活 精子细胞。为此，帕杜博士将构建HSR-omega DNA的融合 以及b2-微管蛋白启动子，它是一种控制元件，专门指导 在雄性生殖系中的基因表达，并将这些构建物插入 苍蝇。然后，她将测试转基因种群的男性生育力。

The current application emphasizes genetic approaches to the study of hsr-omega. It has three specific aims. The first aim is 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 is 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 is 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.