MOLECULAR GENETICS OF HEME BIOSYNTHETIC ENZYMES

本项目的长期目标是剖析监管事件 控制红系成熟过程中血红素的合成 祖细胞，重点是编码第二种酶的基因， 血红素生物合成途径，δ-氨基乙酰丙酸酯酶（ALA-D）。 确定了四个具体目标。 首先，红细胞集落形成单位 （CFU-E）将从甲砜霉素处理的贫血小鼠中纯化， 随后进行培养，以获得来自不同阶段的样品， 红细胞生成 前四种酶和最后一种酶的酶测定 血红素生物合成途径将被执行，以确定 红系细胞中的限速步骤。 同时定量 通过RNA酶保护试验改变mRNA水平， 在转录水平控制限速步骤。 第二，ALA-D基因的红系特异性转录启动子 将使用凝胶阻滞试验和DNaseI进行详细分析 足迹法来定位体外结合蛋白质的核苷酸序列。 第三，在前一节中确定的序列将是 在鼠中通过瞬时转染测定进行功能测试 红白血病细胞和关键序列鉴定的损失 在体外诱变后的功能。 第四， ALA-D基因剂量多态性在近交系小鼠中的发现将被推广 抓到的野生老鼠 复制的分子结构和 将在核苷酸水平上测定和分析三重基因座， 序列水平和模型将建立的机制，增加 通过重组和基因复制表达ALA-D，这可能是 适用于基因组的其他区域。

The long-range goals of this project are to dissect the regulatory events controlling heme biosynthesis during the maturation of erythroid progenitor cells with emphasis on the gene encoding the second enzyme of the heme biosynthetic pathway, delta-aminolevulinate dehydratase (ALA-D). Four specific aims are set. First, erythroid colony-forming units (CFU-E) will be purified from thiamphenicol-treated, anemic mice and subsequently cultured to obtain samples from various stages of erythropoiesis. Enzyme assays for the first four and the last enzyme of the heme biosynthetic pathway will be performed to determine the rate-limiting steps in erythroid cells. Concurrent quantitation of changing mRNA levels by RNase protection assays will identify which of the rate-limiting steps are controlled at the transcriptional level. Second, the erythroid-specific transcriptional promoter of the ALA-D gene will be analyzed in detail using gel-retardation assays and DNaseI footprinting to locate nucleotide sequences which bind proteins in vitro. Third, the sequences identified in the previous section will be functionally tested by transient transfection assays in murine erythroleukemia cells and crucial sequences identified by loss of function following in vitro mutagenesis. Fourth, characterization of the ALA-D gene dosage polymorphism found in the inbred mouse will be extended to wild caught mice. The molecular structure of the duplicated and triplicated loci will be determined and analyzed at the nucleotide sequence level and a model will be built for the mechanism of increased ALA-D expression by recombination and gene duplication which may be applicable to other regions of the genome.