Peroxisome biogenesis and human peroxisome assembly disorders : Approches to prenatal diagnosis and gene therapy.

过氧化物酶体生物合成和人类过氧化物酶体组装障碍：产前诊断和基因治疗方法。

基本信息

批准号：
08557011
负责人：
FUJIKI Yukio
金额：
$ 10.69万
依托单位：
KYUSHU UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1996
资助国家：
日本
起止时间：
1996 至 1998
项目状态：
已结题

项目摘要

With the support of this Grants-in Aid for Scientific Research 0 8 5 5 7 0 1 1 we obtained two major types of findings :I) Isolation, characterization, and complementation group analysis of novel CHO cell mutants defective in peroxisome biogenesisIn addition to the previously isolated, three complementation groups (CGs) of peroxisome-deficient CHO cell mutants, ZP24, Z65, and ZP92, we isolated ZPlO7 (the same group as Z24), ZP1O5/ZP139, ZPIO9, ZP11O, ZP114, ZP119, ZP124, and ZP126, by the P9OH/UV method. CG analysis by PEX cDNA transfection and/or cell fusion with previously identified CGs of mutant cells, including 12 CGs of fibroblasts derived from patients with peroxisome biogenesis disorders (PBDs), revealed that ZP110, ZP114, and ZP126 are distinct from human CGs. Thus, it is evident that peroxisome assembly requires at least 15 gene-products.II) Cloning of novel peroxin genes By genetic functional complementation assay of newly isolated CHO cell mutants, we cloned several peroxin cDNAs (PEXs), including PEX1, PEX12, and PEX19 for ZP1O7, ZP1O9, and ZP119, respectively. We then delineated that gene mutations in PEX1, PEX12, and PEX19 are responsible for PBDs of CG-I (E), CG-III, and CG-J, respectively. Moreover, we isolated PEX1O and PEX16 by expressed sequence tag (EST) DNA search using yeast genes and demonstrated that inactivation of PEX]O and PEX16 is the genetic cause of CG-VII (B) and CG-IX (D) PBDs, respectively. We also found two isoforms of the peroxin Pex5p (PTS 1 receptor) using CG-ll CHO mutants, ZP1O5 and ZP139. Impaired import of PTS1 proteins in the mutant cells was restored by both, shorter and longer, forms of Pex5p. More strikingly, the longer isoform of Pex5p was found to be involved in import of PTS2 proteins as well.

在这项科学研究资助 0 8 5 5 7 0 1 1 的支持下，我们获得了两类主要的发现：I) 过氧化物酶体生物发生缺陷的新型 CHO 细胞突变体的分离、表征和互补组分析除了先前分离的过氧化物酶体缺陷型 CHO 细胞突变体 ZP24、Z65 和 ZP92 的三个互补组 (CG) ZP107(与Z24相同的组)、ZP1O5/ZP139、ZP109、ZP11O、ZP114、ZP119、ZP124和ZP126，通过P9OH/UV方法测定。通过 PEX cDNA 转染和/或与先前鉴定的突变细胞 CG（包括来自过氧化物酶体生物发生障碍 (PBD) 患者的成纤维细胞的 12 个 CG）进行细胞融合进行的 CG 分析表明，ZP110、ZP114 和 ZP126 与人类 CG 不同。因此，很明显，过氧化物酶体的组装需要至少15个基因产物。 II)新型过氧化物酶基因的克隆通过对新分离的CHO细胞突变体的遗传功能互补分析，我们克隆了几个过氧化物酶cDNA（PEX），包括PEX1、PEX12和PEX19 分别用于 ZP1O7、ZP1O9 和 ZP119。然后我们描述了 PEX1、PEX12 和 PEX19 中的基因突变分别导致 CG-I (E)、CG-III 和 CG-J 的 PBD。此外，我们使用酵母基因通过表达序列标签（EST）DNA搜索分离了PEX1O和PEX16，并证明PEX]O和PEX16的失活分别是CG-VII（B）和CG-IX（D）PBD的遗传原因。我们还使用 CG-II CHO 突变体发现了过氧化物酶 Pex5p（PTS 1 受体）的两种亚型，ZP1O5 和 ZP139。突变细胞中受损的 PTS1 蛋白输入可通过较短和较长形式的 Pex5p 恢复。更引人注目的是，发现较长的 Pex5p 亚型也参与 PTS2 蛋白的输入。