MOLECULAR BASIS OF PEROXISOMAL DISORDERS

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The first three years of this award will be carded out under the direction of Dr. David Valle in the Dept of Molecular Biology & Genetics at the Johns Hopkins Univ School of Medicine. His laboratory is extremely well-equipped to provide me with a strong foundation in molecular biology and genetics, which will add to my previous research experience and better enable me to investigate human disease at the molecular level. The last two years of this award will assist me in starting my own laboratory within the Dept of Pediatrics. For this proposal, I will be investigating inborn errors of peroxisomal biogenesis. Peroxisomes are ubiquitous subcellular organelles with a characteristic set of integral membrane proteins and over 40 matrix enzymes which perform a variety of oxidative and synthetic reactions. Several genetic diseases, including Zellweger Syndrome, neonatal adrenoleukodystrophy, and infantile Refsum disease appear to be caused by defects in the biogenesis of peroxisomes. This laboratory is interested in the molecular basis of these inborn errors. Ongoing investigations in the lab are aimed at Determining the normal function and possible involvement of the 70 kD and 35 kD peroxisomal , membrane proteins in these disorders. Human cDNAs for both these proteins have been isolated and characterized. We are utilizing a large collection of cultured fibroblasts from clinically and biochemically well-characterized patients with these disorders (assembled by Dr. H.Moser). Erdman et al (1991) reported the isolation of a S cerevisiae gene (PAS1) capable of rescuing mutants defective for peroxisomal assembly. PAS1 is not a membrane protein and it appears to encode a previously-unidentified protein whose function is necessary for peroxisomal biogenesis. The overall aims of this proposal are to isolate the human homolog of PAS1, determine if it has a similar function in mammalian cells, and determine if mutations in this gene may be responsible for one subset of defects in peroxisomal biogenesis. The specific aims are: to isolate a cDNA clone encoding human PAS1 either by use of degenerate oligonucleotides or by functional complementation in yeast; to determine the nucleotide sequence of the full-length human PAS1 CDNA and to assess its ability to function in yeast; to delineate the organization of the human PAS1 gene and determine its chromosomal localization; and to identity and characterize mutations in the human PAS1 gene. in patients with inborn errors of peroxisomal biogenesis.