MOLECULAR GENETICS OF HEME BIOSYNTHETIC ENZYMES

这个项目的长期目标是剖析监管事件 红系成熟过程中血红素生物合成的调控 祖细胞，重点是编码第二种酶的基因 血红素的生物合成途径，三角洲-氨基酮戊酸脱水酶(ALA-D)。 制定了四个具体目标。第一，红系集落形成单位 (CFU-E)将从甲氧西林治疗的贫血小鼠和 随后进行培养以从不同阶段获得样本 红血球生成。前四种酶和最后一种酶的酶分析 将进行血红素生物合成途径以确定 红系细胞的限速步骤。并发量化 通过核糖核酸酶保护试验改变信使核糖核酸水平将确定 限速步骤在转录水平上受到控制。 第二，ALA-D基因的红系特异性转录启动子 将使用凝胶延迟分析和DNaseI进行详细分析 在体外定位结合蛋白质的核苷酸序列的足迹。 第三，上一节中确定的序列将是 用瞬时转染法检测小鼠的功能 红白血病细胞和关键序列的丢失 体外诱变后的功能。第四，人物塑造 在近交系小鼠中发现的ALA-D基因剂量多态性将被扩大 去野捕老鼠。复制和复制的分子的分子结构 将在核苷酸上确定和分析三个重复的基因座 并建立了序列层次的递增机理模型 ALA-D通过重组和基因复制表达，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.