MOLECULAR BASIS OF PEROXISOMAL DISORDERS

该奖项的前三年将在指导下进行 大卫瓦莱博士在分子生物学和遗传学系在约翰 霍普金斯大学医学院。 他的实验室设备极其齐全 为我提供了分子生物学和遗传学的坚实基础， 这将增加我以前的研究经验，使我能够更好地 在分子水平上研究人类疾病 的最后两年 这个奖项将帮助我在部门内开始我自己的实验室， 儿科. 对于这个建议，我将调查先天性的错误， 过氧化物酶体生物发生 过氧化物酶体是普遍存在的亚细胞器 具有一组特征性的完整膜蛋白和超过40种基质 进行各种氧化和合成反应的酶。 几种遗传性疾病，包括齐薇格综合征，新生儿 肾上腺脑白质营养不良和婴儿Refsum病似乎是由 过氧化物酶体生物发生的缺陷。 该实验室感兴趣的是 这些先天缺陷的分子基础 正在进行的调查 实验室旨在确定正常功能和可能的参与 70 kD和35 kD过氧化物酶体，膜蛋白在这些疾病。 这两种蛋白质的人cDNA已被分离和表征。 我们正在利用大量的培养成纤维细胞从临床 并且生化特征良好的患有这些疾病的患者 （由H.Moser博士整理）。Erdman等（1991）报道了分离到的一种 酿酒酵母基因（PAS 1）能够拯救突变体缺陷， 过氧化物酶体装配 PAS 1不是一种膜蛋白， 编码一种以前未鉴定的蛋白质，其功能是必需的， 过氧化物酶体生物发生 该提案的总体目标是隔离 PAS 1的人类同源物，确定它是否具有类似的功能， 哺乳动物细胞，并确定该基因的突变是否可能导致 过氧化物酶体生物发生缺陷的一个子集。 具体目标 分离编码人PAS 1的cDNA克隆， 简并寡核苷酸或通过酵母中的功能互补; 确定全长人PAS 1 cDNA的核苷酸序列，并 评估其在酵母中发挥作用的能力;描绘组织 人PAS 1基因并确定其染色体定位;以及 鉴定和表征人PAS 1基因中的突变。患者 有过氧化物酶体生物合成的先天缺陷

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.