GENE REGULATION IN DROSOPHILA: PTERIDINE

这个项目的目的是为了研究组织和 黑腹金丝猴Punch(Pu)基因座的调控轨迹 编码酶，GTP环水解酶，该酶催化 蝶啶生物合成中的第一个反应。蝶呤类化合物可用作 果蝇体内的色素，但也被普遍用作酶 包括5-羟色胺和5-羟色胺合成的反应的辅助因子 儿茶酚胺。人类体内的蝶呤缺乏是致命的。PU是 组织和发展表达上的复杂性，如 由发育特异性突变的产生来指示 以及复杂的等位基因互补模式。我们的 研究表明，Pu至少编码三种多肽， 每一种基因都在不同的发育时期表达。 他们各自的表达时间对应于知道的时间段 通过酶分析或突变表型来评估PU的功能。 补充致死性和发育特异性的Pu突变 有相应的离散的和可预测的蛋白质变化 到特定的等位基因互补组。每一个都是致命的 互补组也对应于可预测的，以及 本质上相互排斥的胚胎致死表型， 影响胚胎晚期致死表型的一组等位基因， 一组中的等位基因影响晚期胚胎功能 另一个组的等位基因影响很早的模式 功能。因此，我们发现了两者之间的直接关联 基因座、蛋白质产物和突变体的遗传域 表型。拟议中的实验旨在阐明 关于这个基因座复杂性的分子基础，以及 确定钚的调节机制。大约200人 已经克隆了Pu区DNA的Kb，并对其进行了研究 该区域的实体组织已经开始。这些实验 在这个提案中描述的是分子的延续 基因分析正在进行中。分子作图 与Pu相关的重新排列、删除和插入 突变和邻近互补中的突变 分组将完成。成绩单将由Northern绘制 印迹、S1核酸内切酶降解和引物延伸 实验。正在筛选和分离cDNA文库 将对克隆的cdna进行鉴定。杂交选育--试管苗 翻译经验和获取更具体信息的方法 针对Pu产品的抗体正在计划中。普通话成绩单 将检测突变体，并进行单基因原位杂交。 为了评估Pu，建议在组织切片上使用链式探针 在发育时期和其他组织中的表达 分析的形式是不实际的。

The purpose of this project is to stuey the organization and regulation of the Punch (Pu) locus in D. melanogaster. The locus encodes the enzyme, GTP cyclohydrolase, the enzyme catalyzing the first reaction in pteridine biosynthesis. Pterins serve as pigments in Drosophila, but also are universally used as enzyme cofactors for reactions that include the synthesis of serotonin and catecholamines. Pterin deficiencies in humans are lethal. Pu is complex in its organization and development expression, as indicated by the generation fo developmentally-specific mutations and by complex patterns of interallelic complementation. Our research suggests that Pu encodes at least three polypeptides, each of which is expressed at a discrete developmental time. Their individual times of expression correspond to periods of know Pu function as assessed by enzyme assays or mutant phenotypes. Complementing lethal and developmentally specific Pu mutations have discrete and predictabfle protein alterations taht correspond to specific interallelic complementation groups. Each lethal complementation groups also corresponds to predictable, and essentially mutually exclusive embryonic lethal phenotypes, with alleles in one group affecting late embryonic lethal phenotypes, with alleles in one group affecting late embryonic functions and alleles of another group affecting very early, patterning functions. Thus, we have discovered direct correlations between genetic domains of the locus, protein products and mutant phenotypes. The proposed experiments are intended to shed light on the molecular basis for the complexity of this locus, and to determine the mechanisms by which Pu is regulated. About 200 kb of the Pu region DNA have been cloned, and a study of the physical organization of the region has begun. The experiments described in this proposal are a continuation of the molecular genetic analysis now in progress. Molecular mapping of rearrangements, deletions and insertions associated with Pu mutations and with mutations in neighboring complementation groups will be completed. Transcripts will be mapped by Northern blot, S1 endonuclease degestion, and primer extension experiments. Libraries of cDNAs are being screened, and isolated cDNA clones will be characterized. Hybrid selection- in vitro translation experiemnts and means of obtaining more specific antibodies to the Pu products are planned. Transcripts in Pu mutants will examined, and in situ hybridization of single- stranded probes to tissue sections is proposed in order to assess Pu expression in developmental times and in tissues where other forms of analysis are not practical.