STUDIES OF PORPHYRIA AND HUMAN HEME BIOSYNTHESIS

卟啉症和人血红素生物合成的研究

• 批准号: 3228048
• 负责人: Robert J Desnick
• 金额: $ 29.21万
• 依托单位: MOUNT SINAI SCHOOL OF MEDICINE OF CUNY
• 依托单位国家: 美国
• 财政年份: 1980
• 资助国家: 美国
• 起止时间: 1980-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3228048
• 关键词: RNA splicing; Retroviridae; affinity chromatography; autosomal dominant trait; biological polymorphism; bone marrow transplantation; cats; chromosomes; clone cells; complementary DNA; congenital erythropoietic porphyria; congenital hepatic porphyria; decarboxylases; disease /disorder model; enzyme biosynthesis; enzyme linked immunosorbent assay; enzyme mechanism; enzyme structure; gene expression; gene mutation; gene therapy; genetic disorder diagnosis; genetic manipulation; genetic mapping; heme; heterozygote; high performance liquid chromatography; human genetic material tag; human tissue; immunoelectrophoresis; immunohematology; in situ hybridization; isozymes; laboratory mouse; laboratory rabbit; molecular cloning; molecular genetics; molecular pathology; monoclonal antibody; nucleic acid hybridization; nucleic acid probes; nucleic acid sequence; orphan disease /drug; porphobilinogen; porphobilinogen synthase; porphyrin biosynthesis; protein engineering; structural genes; transcription factor; transfection

The overall objective of the proposed research is to investigate the inborn errors and molecular genetics of the four human cytosolic heme biosynthetic enzymes, delta-aminolevulinic acid dehydratase (ALA-D), hydroxymethylbilane synthase (HMB-S), uroporphyrinogen III synthase (URO-S) and uroporphyrinogen decarboxylase (URO-D). Using the full-length hepatic cDNAs for ALA-D, HMB-S, URO-S and URO-D which have been cloned and sequenced in this laboratory, their respective full-length erythroid cDNAs will be isolated and characterized for identification of tissue-specific sequences in their 5' or 3' regions, determination of their respective cap sites and for prokaryotic expression of the active enzymes. Sub-chromosomal regional localization for HMB- S and URO-S will be assigned by somatic cell and in situ hybridization. Genomic sequences encoding all four enzymes will be isolated; the 5' and 3' regions as well as the intron-exon boundaries will be sequenced and compared. For studies of transcription, chimeric constructs of the genomic 5'-flanking sequences of each gene and the bacterial chloramphenicol acetyltransferase gene will be transfected into K562 erythroid and HepG2 hepatic cells. Deletions and substitution mutations in specific 5' regions and DNase I protection experiments with hepatic and erythroid nuclear extracts will be performed to identify transcriptional regulatory elements and nuclear factors involved in the tissue-specific expression of these genes. The nature of the molecular lesions in unrelated patients with ALA-D deficiency, acute intermittent porphyria (AIP) and cogenital erythropoietic porphyria (CEP) will be determined. Efforts will be directed to identify informative RFLPs for the precise diagnosis of heterozygotes for AIP and porphyria cutanea tarda (PCT. The biochemical and molecular abnormalities in cats with HMB-S dificiency will be characterized. This animal model will permit the unique opportunity to evaluate the metabolic effects of therapeutic modalities, including bone marrow transplantation and retroviral-mediated gene transfer. These studies should provide 1) increased understanding of the structure, organization and tissue-specific regulation of these heme biosynthetic enzymes, 2) delineation of the molecular lesions in these disorders as well as 3) new methods for the precise diagnosis and potential therapy of these inborn errors of heme biosynthess.

拟议研究的总体目标是调查 四个人类的先天错误和分子遗传学 胞质血红素生物合成酶、δ-氨基乙酰丙酸 脱水酶（ALA-D）、羟甲基联烷合酶（HMB-S）、 尿卟啉原 III 合酶 (URO-S) 和尿卟啉原 脱羧酶（URO-D）。 使用全长肝 cDNA ALA-D、HMB-S、URO-S 和 URO-D 已被克隆并 在本实验室测序后，它们各自的全长红细胞 cDNA 将被分离并表征以鉴定 5' 或 3' 区域的组织特异性序列，测定 它们各自的帽位点和原核表达 活性酶。 HMB-的亚染色体区域定位 S和URO-S将由体细胞原位分配 杂交。 编码所有四种酶的基因组序列将 被孤立； 5' 和 3' 区域以及内含子-外显子 边界将被排序和比较。 用于研究 转录，基因组 5' 侧翼的嵌合构建体 每个基因和细菌氯霉素的序列 乙酰转移酶基因将转染至K562红细胞 和HepG2肝细胞。 缺失和取代突变 特定 5' 区域和 DNase I 保护实验 将进行肝和红细胞核提取物 识别转录调控元件和核因子 参与这些基因的组织特异性表达。 这 无关的 ALA-D 患者的分子病变的性质 缺乏症、急性间歇性卟啉症（AIP）和先天性 将测定红细胞生成性卟啉症 (CEP)。 努力将会 指导识别信息丰富的 RFLP，以实现精确的 AIP 和迟发性皮肤卟啉症杂合子的诊断 （PCT。猫的生化和分子异常 HMB-S 缺陷将被表征。 该动物模型将 提供评估代谢影响的独特机会 治疗方式，包括骨髓移植 和逆转录病毒介导的基因转移。 这些研究应该 提供 1) 增加对结构、组织的理解 以及这些血红素生物合成的组织特异性调节 酶，2) 描绘这些疾病中的分子病变 以及3）精确诊断的新方法和潜力 治疗这些血红素生物合成的先天性错误。