ONTOGENY AND PHYLOGENY OF HUMAN PEROXISOMAL FUNCTION

尽管人类出生缺陷和综合征的频率，一些原则， 用来解释它们的胚胎发生改变。 人类 过氧化物酶体紊乱提供了一种生物化学方法来理解 一组综合征影响眼，耳，脑，肝，肾， 骨头 齐薇格综合征，这些疾病的原型，是一个 常染色体隐性遗传病，其中过氧化物酶体，但不是所有的过氧化物酶体 大多数组织中不存在蛋白质。 这一长期目标 建议是定义齐薇格综合征的基本缺陷， 构建可用于研究其 发病机制和治疗 该方法将侧重于以下组成部分： 过氧化物酶体，可以很容易地纯化，完整的过氧化物酶体 膜蛋白或PMP。 145、70、54、36和58的至少5种不同的PMP， 22 Kd可通过蛋白电泳或免疫印迹法可视化， 在大鼠、小鼠和人类中高度保守。最初的重点将是 在PMP 22上，因为可以获得用于 这种蛋白质包括氨基末端区域。 人和啮齿动物 肝cDNA文库可用于cDNA克隆的分离 使用针对小鼠PMPs的多特异性兔抗体， 或基于部分氨基酸序列的寡核苷酸混合物。 分离PMP 22 cDNA克隆，然后进行完全双脱氧 对两条链进行测序并通过比较确认克隆同一性 与氨基酸序列。 该cDNA序列将成为 比较大鼠、小鼠和人的PMP 22 cDNA序列， MOPAC版聚合酶链反应。 特征化cDNA 序列也将用于基因组PMP的初步Southern分析 三种生物体中有22个序列。 将检查人PMP 22基因 对于RFLPs和cDNA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，假定的羧基末端靶向信号-将被 检查以深入了解PMP 22的功能。 时空表达 还将检查小鼠/人个体发育期间PMP 22的表达，以获得 对过氧化物酶体生物发生的理解 负责齐薇格综合征的基因将被探索， 候选基因（PMP 22）的方法和尝试，以补充人类 齐薇格综合征或酵母过氧化物酶体=组装缺陷细胞。

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.