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

这对于科学研究基金援助的支持下0 8 5 5 0 1 1 7我们获得两大类型的研究:1)分离、鉴定,和互补群分析小说CHO细胞突变体的缺陷在过氧物酶体biogenesisIn先前孤立之外,三个互补组(CGs) peroxisome-deficient CHO细胞突变体,ZP24, Z65, ZP92,我们孤立ZPlO7(同一组Z24), ZP1O5 / ZP139 ZPIO9, ZP11O, ZP114, ZP119, ZP124, ZP126,P9OH/UV法。通过转染PEX cDNA和/或与先前鉴定的突变细胞CG进行细胞融合分析，包括来自过氧化物酶体生物发生障碍（PBDs）患者的12个成纤维细胞CG，发现ZP110、ZP114和ZP126与人类CG不同。因此，很明显，过氧化物酶体的组装至少需要15个基因产物。通过对新分离的CHO细胞突变体的遗传功能互补实验，我们分别克隆了zp107、zp109和ZP119的过氧化物基因PEX1、PEX12和PEX19。然后，我们描述了PEX1、PEX12和PEX19基因突变分别与CG-I (E)、CG-III和CG-J的pbd有关。此外，我们利用酵母基因通过表达序列标记（EST） DNA搜索分离出pex10和PEX16，并证明PEX]O和PEX16的失活分别是导致CG-VII (B)和CG-IX (D) pbd的遗传原因。我们还使用CG-ll CHO突变体zp105和ZP139发现了过氧化物Pex5p （PTS 1受体）的两个同工型。突变细胞中受损的PTS1蛋白输入通过较短和较长形式的Pex5p得以恢复。更引人注目的是，Pex5p的长同种异构体也被发现参与了PTS2蛋白的进口。

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.