MOLECULAR GENETICS OF HEME BIOSYNTHETIC ENZYMES

关于血红素代谢及其扰动，我们知道得很多， 这一重要物质的产生的分子机制 材料是规范的。 我们的目标是描绘这样的机制， 关于血红素生物合成中前两种酶的表达， 途径：δ-氨基乙酰丙酸合成酶（ALAS）和δ-氨基乙酰丙酸 酶（ALAD）。 为了这项事业，我们带来了经过验证的老鼠肝脏 ALAD-cDNA克隆及抗大鼠肝ALAD单克隆抗体的研制 和阿拉斯。 设定了七个具体目标：第一，隔离， 全长小鼠和大鼠ALAD-cDNA的表征;第二，分离 以及两个物种中ALAD基因组序列的描绘;第三， 证实了我们的发现，ALAD基因剂量的差异帐户 对于近交系小鼠之间ALAD酶活性的增量差异， 第四，评估（a）ALAD基因拷贝的遗传多态性 异交小鼠和野生小鼠中的数量，以及（B）其与 环境铅;第五，ALAD的分子性质的描绘， 非典型的设置，例如，红白血病K562细胞，胎儿 第六，分离全长ALAS-cDNA， 从大鼠和小鼠肝脏中提取相应的基因DNA;第七、应用 ALAS-和ALAD-探针的分子机制解剖 在鼠红白血病细胞中酶表达的快速流动的基础， 在小鼠红系祖细胞中，以及在用 卟啉生成剂。

Much is known about heme metabolism and its perturbations, little is known of the molecular mechanisms by which the production of this essential material is regulated. Our goals are to delineate such mechanisms with respect to the expression of the first two enzymes in the heme biosynthetic pathway: delta-aminolevulinate synthase (ALAS) and delta-aminolevulinate dehydrase (ALAD). To this undertaking, we bring verified rat liver ALAD-cDNA clones and monoclonal antibodies developed against rat liver ALAD and ALAS. Seven specific aims are set: first, isolation and characterization of full-length mouse and rat ALAD-cDNA; second, isolation and delineation of ALAD genomic sequences in both species; third, verification of our finding that differences in ALAD-gene dosage account for incremental differences in ALAD enzymatic activity between inbred mouse strains; fourth, assessment of (a) genetic polymorphism for ALAD-gene copy number in outcrossed and feral mice and (b) its possible relationship to environmental lead; fifth, delineation of the molecular nature of ALAD in settings where it is atypical, e.g., erythroleukemia K562 cells, fetal liver and human mutants; sixth, isolation of full-length ALAS-cDNA and corresponding gene DNA from rat and mouse liver; and seventh, application of ALAS- and ALAD-probes to the dissection of molecular mechanisms underlying rapid flux of enzyme expression in murine erythroleukemia cells, in murine erythroid progenitors, and in the livers of animals treated with porphyrinogenic agents.