Structure and function of the regulatory region of the gene encoding non-specific form delta-aminolevulinate synthase

血红素是所有真核细胞所必需的，因为它是许多蛋白质的修复基团，其细胞内浓度受到高度调控。在动物体内，血红素生物合成的第一步也是限速步骤是由β-氨基乙酰丙酸(ALA)合成酶催化的。ALA合成酶有两种组织特异性同工酶：红系特异性ALA合成酶(ALAS-E)和非特异性ALA合成酶(ALAS-N)。为了了解控制血红素生物合成途径的分子机制，首先要分析ALA合成酶基因的结构和功能。我们试图分离和鉴定编码大鼠ALAS-N的基因。ALAS-N基因在大鼠基因组中跨度超过14kb，包括11个外显子，对该基因启动子区域的分析揭示了几个潜在的协同作用调控元件。比较大鼠ALAS-N基因和小鼠ALAS-E基因的组织结构表明，动物ALA合成酶的祖先基因可能由11个外显子组成，并且ALAS-N和ALAS-E基因都是由该祖先基因衍生而来的。结果表明，红系转录因子的结合位点聚集在转录起始点上游120bp的范围内。采用RNA印迹杂交技术检测了胚胎、新生和成年大鼠肝脏中ALAS-N和ALAS-E基因的发育期特异性转录。结果表明，虽然ALAS-E是为合成血红蛋白提供大量血红素的关键酶，但ALAS-N的功能是向肝脏中的P-450系统供应血红素，同时也是管家基因，为各种组织中的呼吸细胞色素和其他血红蛋白提供血红素。

Heme is essential for all eukaryotic cells because of its role as a prosthetic group for a number of proteins, and its intracellular concentration is highly regulated. The first and the rate-limiting step of heme biosynthesis in animals is catalyzed by delta-aminolevulinate(ALA) synthase. There are two tissue-specific isozymes of ALA synthase : the erythroid- specific ALA synthase (ALAS-E) and the non-specific ALA synthase (ALAS-N). To understand the molecular mechanisms contorolling the heme biosynthetic pathway, it is a prerequisite to analyze the structure and function of the ALA synthase genes.We attempted isolation and characterization of the gene encoding rat ALAS-N.The ALAS-N gene was found to span more than 14kb in the rat genome, encompassing 11 exons, Analysis of the promoter region of the gene revealed several potential cos-acting regulatory elements. Comparison of the organization of rat ALAS-N gene with that of the ALAS-E gene in mouse suggests that the ancestral gene for ALA synthase in animals was probably composed of 11 exons, and both ALAS-N and ALAS-E genes were derived from this ancestral gene.The proximal regulatory region of the human ALAS-E gene was also analyzed. The results indicated that binding sites for erythroid transcription factors are clustered within 120 bp upstream form the transcription initiation site.RNA blot hybridization analysis was used to examine the developmental stage-specific transcription of ALAS-N and ALAS-E genes in fetal, newborn and adult rat liver. The results demonstrated that, while ALAS-E is the key enzyme which supplies large quantities of heme for hemoglobin synthesis, ALAS-N functions to supply heme to the P-450 system in the liver and also functions as a housekeeper gene to supply heme for respiratory cytochromes and other hemoproteins in various tissues.