GENE REGULATION IN DROSOPHILA: PTERIDINE

这个项目的目的是stuey组织和 调控D.黑腹菌 基因座 编码GTP环化水解酶， 是蝶啶生物合成的第一步反应。 蝶呤作为 色素，但也普遍用作酶 反应的辅助因子，包括血清素的合成， 儿茶酚胺 人类缺乏蝶呤是致命的。 Pu是 复杂的组织和发展表达，如 由发育特异性突变的产生指示， 和复杂的等位基因间互补模式。 我们 研究表明Pu编码至少三种多肽， 每一种都在不连续的发育时间表达。 它们各自的表达时间对应着认知的时期 通过酶测定或突变表型评估钚功能。 补充致命和发育特异性Pu突变 具有离散的和可预测的蛋白质改变， 与特定的等位基因间互补群有关。 每个致命的 互补组也对应于可预测的， 基本上相互排斥的胚胎致死表型， 一组中的等位基因影响晚期胚胎致死表型， 一组中的等位基因影响晚期胚胎功能， 另一组等位基因影响极早期，模式化 功能协调发展的 因此，我们已经发现， 基因座的遗传结构域、蛋白质产物和突变体 表型 拟议中的实验旨在阐明 在这个基因座的复杂性的分子基础上， 确定钚的调节机制。 约200 已克隆了Pu区DNA的2.5kb， 该地区的实际组织工作已经开始。 实验 在这个建议中描述的是分子的延续， 基因分析正在进行中。 的分子定位 与Pu相关的重排、缺失和插入 突变和相邻互补突变 组将完成。 成绩单将由北方 印迹、S1内切酶消化和引物延伸 实验 cDNA文库正在筛选和分离 将表征cDNA克隆。 杂种选择-体外 翻译经验和方法，获得更具体的 计划对钚产品进行抗体测试。 成绩单在Pu 突变体将检查，并在原位杂交的单- 为了评估钚，提出了一种用于组织切片的单链探针 在发育时期和组织中表达， 分析方法不实用。

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.