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.
NIH ExPorter
NIH ExPorter