ONTOGENY AND PHYLOGENY OF HUMAN PEROXISOMAL FUNCTION

人类过氧化物酶体功能的个体发生和系统发育

• 批准号: 3327744
• 负责人: GOLDER N WILSON
• 金额: $ 11.55万
• 依托单位: UT SOUTHWESTERN MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3327744
• 关键词: cerebrohepatorenal syndrome; complementary DNA; developmental genetics; embryo /fetus culture; evolution; gene complementation; genetic library; genetic mapping; histogenesis; human genetic material tag; human tissue; laboratory mouse; laboratory rabbit; laboratory rat; membrane proteins; natural gene amplification; peroxisome; polymerase chain reaction; protein sequence; protein structure function; restriction fragment length polymorphism; southern blotting; western blottings

Despite the frequency of human birth defects and syndromes, few principles have been defined to explain their altered embryogenesis. Human peroxisomal disorders provide a biochemical approach to the understanding of one group of syndromes affecting eye, ear, brain, liver, kidney, and bone. Zellweger syndrome, the prototype of these disorders, is an autosomal recessive disease where peroxisomes, but not all peroxisomal proteins, are absent from most tissues. The long term objective of this proposal is to define the basic defect(s) in Zellweger syndrome and to construct cellular and animal models which can be used to investigate its pathogenesis and therapy. The approach will focus on a component of peroxisomes which can be readily purified, the integral peroxisomal membrane proteins or PMP. At least 5 distinct PMPs of 145, 70, 54, 36, and 22 Kd can be visualized by protein electrophoresis or immunoblotting which are highly conserved among rat, mouse, and man. The initial focus will be on PMP 22 because of the availability of a partial amino acid sequence for this protein which include the amino-terminal region. Human and rodent liver cDNA libraries are available for the isolation of cDNA clones corresponding to PMP 22 using polyspecific rabbit antibodies to mouse PMPs or oligonucleotide mixtures based on partial amino acid sequences. Isolation of a PMP 22 cDNA clone will be followed by complete dideoxy sequencing of both strands and confirmation of clone identity by comparison with amino acid sequences. This cDNA sequence will be the basis for comparative sequencing of PMP 22 cDNAs from rat, mouse and man using the MOPAC version of the polymerase chain reaction. Characterized cDNA sequences will also be used for prelimary Southern analysis of genomic PMP 22 sequences in the three organisms. Human PMP 22 genes will be examined for RFLPs and interesting regions of both cDNA and genomic sequences--upstream flanking, transcription initiation, N-terminal coding, intron/ exon junctions, putative carboxyterminal targeting signals--will be examined to gain insights into PMP 22 function. Spatiotemporal expression of PMP 22 during mouse/ human ontogeny will also be examined to gain understanding about peroxisome biogenesis Two strategies for identifying the gene responsible for Zellweger syndrome will be explored consisting of the candidate gene (PMP 22) approach and an attempt to complement human Zellweger syndrome or yeast peroxisome=assembly-deficient cells.

尽管人类出生缺陷和综合症的频率很高，但很少有原则 已经被定义为解释它们改变的胚胎发生。人类 过氧化物体疾病提供了一种生物化学方法来理解 一组影响眼、耳、脑、肝、肾、肾的证候 骨头。齐薇格综合征是这些疾病的原型，是一种 常染色体隐性遗传病中的过氧体，但不是所有的过氧体 蛋白质，在大多数组织中都不存在。这样做的长期目标是 建议对Zellweger综合征的基本缺陷(S)进行界定，并 构建细胞和动物模型，可用于研究其 发病机制及治疗。该方法将侧重于以下组件 容易提纯的过氧化物体，完整的过氧化物体 膜蛋白或PMP。至少5个不同的PMP，分别为145、70、54、36和 22kD可通过蛋白质电泳法或免疫印迹法显示 在老鼠、小鼠和人中高度保守。最初的重点将是 在PMP 22上，因为有部分氨基酸序列可用于 这种蛋白质包括氨基末端区域。人和啮齿动物 肝脏c DNA文库可用于分离c DNA克隆 对应于PMP 22的多特异性兔抗体用于小鼠PMP 或基于部分氨基酸序列的寡核苷酸混合物。 分离PMP 22的cDNA克隆之后将进行完全的双脱氧 双链测序及比对确认克隆同源性 带有氨基酸序列。这一cdna序列将成为 大鼠、小鼠和人PMP 22基因的比较测序 MOPAC版的聚合酶链式反应。鉴定的基因 序列也将用于基因组PMP的初步Southern分析 三种生物中的22个序列。人类PMP 22基因将被检测 对于RFLP以及cDNAs和基因组的感兴趣区域 序列--上游侧翼，转录起始，N-末端编码， 内含子/外显子连接，推测的羧基末端靶向信号-将是 研究以深入了解PMP 22的功能。时空表达 还将检测小鼠/人类个体发育过程中PMP 22的表达，以获得 对过氧化物体生物发生的认识两种鉴定策略 将探索与齐薇格综合征有关的基因，包括 候选基因(PMP 22)方法和对人类补体的尝试 Zellweger综合征或酵母过氧化酶体=组装缺陷细胞。