We were unable to detect peroxisomal or vesicular staining for these PMPs in PBD061 cells, even though all three proteins were efficiently targeted to peroxisome membranes in PBD094 cells (Fig

We were unable to detect peroxisomal or vesicular staining for these PMPs in PBD061 cells, even though all three proteins were efficiently targeted to peroxisome membranes in PBD094 cells (Fig. find that expression of restores the formation of new peroxisomes in PBD061 cells. Peroxisome synthesis and peroxisomal membrane protein import could be detected within 2C3 h of injection and was followed by matrix protein import. These results demonstrate that peroxisomes do not necessarily arise from division of preexisting peroxisomes. We propose that peroxisomes may form by either of two pathways: one that involves PEX11-mediated division of preexisting peroxisomes, and another that involves PEX16-mediated formation of peroxisomes in the absence of preexisting peroxisomes. alleles, and lacks detectable levels of mRNA and protein (Reuber et al., 1997). The CG2 cell line, PBD005, is homozygous for an inactivating mutation in mRNA and protein (Dodt et al., 1995). The CG3 cell line, PBD097, is a compound heterozygote for two inactivating frameshift mutations in (Chang et al., 1997). The CG4 cell line, PBD105, is also a compound heterozygote for two inactivating frameshift mutations, except in the gene (Yahraus et al., 1996). The CG7 cell line, PBD100, is homozygous for a splice donor site mutation in and expresses a mRNA with a large internal deletion CD163 that lacks activity (Warren et al., 1998). The gene that is mutated in Minoxidil (U-10858) cells from complementation group 8 of the PBDs is unknown but the CG8 cell line used in this study, PBD109, was equally or more severely deficient in peroxisomal matrix protein import than any other CG8 cell line. The CG9 cell line, PBD061, is equivalent to GM6231, a Zellweger syndrome cell line from the Coriell Cell Repository. The CG10 cell line, Minoxidil (U-10858) PBD094, is homozygous for an inactivating nonsense mutation in the gene (Shimozawa et al., 1992). The CG13 cell line, PBD222, is the sole representative of this complementation group and was derived from a neonatal adrenoleukodystrophy patient. Again, the gene defective in this patient remains to be determined. Aside from PBD222, all cell lines used in this study were derived from severely affected Zellweger syndrome patients. Antibodies to PMP70 were obtained from Gerardo Jimenez-Sanchez and David Valle (The Johns Hopkins University School of Medicine, Baltimore, MD). Antibodies to P70R were obtained from Wilhelm Just (University of Heidelberg, Heidelberg, Germany). mAbs to the myc epitope tag were obtained from the tissue culture supernatant of the hybridoma 1-9E10 (Evan et al., 1985). To generate antibodies to PEX16, we first expressed amino acids 145C336 of in fusion with the maltose binding protein. The resulting maltose binding proteinCfusion was purified by affinity chromatography on an amylose resin according to the manufacturer’s instructions (fluorescence microscope and all images were collected on film (T-Maxx 400; gene were identified by searching the database of expressed sequence tags using the TBLASTN algorithm. A mouse cDNA capable of encoding a protein similar to PEX16 was identified. The sequence of the murine cDNA was used to identify a human cDNA by BLAST searches of the human database of expressed sequence tags. Clones encoding the human cDNA were obtained from Genome Systems Inc. and sequenced in their entirety. The ORF was also amplified from a full-length cDNA clone using The insert in this plasmid was sequenced in its entirety to Minoxidil (U-10858) ensure that no mutations were introduced during the cloning process. The cDNA and deduced protein sequences for human are available from GenBank (accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”AF118240″,”term_id”:”4545263″,”term_text”:”AF118240″AF118240). Mutation detection was performed initially by RT-PCR. RNA was extracted from PBD061 cells and from a control human fibroblast and converted to cDNA as follows. Approximately 1 g of total RNA from an unaffected individual and from PBD061 was used as template in a cDNA synthesis reaction using a cDNA. A product of the correct size was obtained from each reaction. However, the yield of cDNA from PBD061 RNA was significantly lower than that obtained from control RNAs, a result that was consistently observed in multiple experiments. The cDNAs from the control group, and PBD061 cells were sequenced and within their entirety directly. The control fragment matched up the cDNA series exactly. On the other hand, the cDNA from PBD061 acquired a 1-bp substitution where the initial nucleotide from the arginine 176 codon, CGA, was changed into a T, terminating the ORF about halfway through the coding area (data not proven). The series chromatograph from the cDNA from PBD061 demonstrated.