PEROXISOME BIOGENESIS--A GENETIC APPROACH

过氧化物酶体生物合成——一种遗传学方法

• 批准号: 2143160
• 负责人: James Michael Cregg
• 金额: $ 12.45万
• 依托单位: OREGON GRADUATE INSTITUTE SCIENCE & TECH
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 1997-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2143160
• 关键词: cytogenetics; developmental genetics; electron microscopy; enzyme activity; fluorescence microscopy; fungal genetics; gene complementation; genetic regulation; membrane proteins; molecular cloning; northern blottings; nucleic acid sequence; peroxisome; phenotype; protein biosynthesis; protein sequence; protein structure function; southern blotting; yeasts

Eukaryotic cells possess a variety of organelles, each devoted to performing a specific set of metabolic functions. The cell must maintain each of these organelles and correctly direct specific sets of proteins to their proper subcellular locations. How the cell manages these operations for some organelles such as the mitochondrion, chloroplast and endoplasmic reticulum has been the subject of intensive investigations. Relative to these, little is known about peroxisomes, a family of ubiquitous organelles, surrounded by a single membrane and containing enzymes involved in a variety of important metabolic pathways. In humans, peroxisomal enzymes are particularly important in lipid metabolism (e.g., plasmalogen and bile acid synthesis; fatty acid, cholesterol and prostaglandin degradation) and genetic. defects in the organelles result in a lethal human disorder, termed Zellweger syndrome. The primary long-term goal of this program is to understand, at the molecular level, the mechanisms which control peroxisome biogenesis and function. The objective of the program is to initiate a genetic approach toward this end using the methanol-utilizing yeast Pichia pastoris as a model. This yeast was selected because peroxisomes are absolutely required for its growth on methanol, an easily observed phenotype, and because methods for classical- and molecular-genetic manipulation of the organism are fully developed. A comprehensive collection of peroxisome-deficient (per) mutants of P. pastoris will be isolated and subjected to genetic, biochemical and cellular biological studies aimed at elucidating the molecular basis of their primary defects. The per mutants will then be utilized to clone the specific genes affected and the DNA sequence of each PER gene will be determined and analyzed. The amino acid sequence deduced from each gene along with the biochemical analysis of the effect of mutations in each gene should help to elucidate the role of each protein in peroxisome function and to formulate an overall picture of peroxisomal operations. A second long-term goal of this program is to utilize knowledge gained from this research to understand the human disease state and to aid Zellweger victims, specifically. It is expected that some of the yeast peroxisomal genes that are identified will be homologues of genes that are affected in Zellweger patients. If so, information obtained on yeast PER genes and their products could be useful in identifying and isolating the human homologues. These human PER genes would be valuable as diagnostic probes and eventually may be used in gene therapy treatments.

真核细胞具有多种细胞器，每种细胞器都致力于 执行特定的代谢功能。 细胞必须保持 这些细胞器中的每一个都能正确地引导特定的蛋白质组 它们的亚细胞位置。 细胞如何管理这些 一些细胞器如叶绿体、叶绿体和 内质网是深入研究的对象。 相对于这些，很少有人知道过氧化物酶体，一个家庭的 普遍存在的细胞器，由一个单一的膜包围，并含有 参与多种重要代谢途径的酶。 在 在人类中，过氧化物酶体酶在脂质中特别重要， 代谢（例如，缩醛磷脂和胆汁酸合成;脂肪酸， 胆固醇和前列腺素降解）和遗传。缺陷 细胞器导致一种致命的人类疾病，称为齐薇格综合征。 该计划的主要长期目标是了解，在 分子水平上，控制过氧化物酶体生物合成的机制， 功能 该计划的目标是启动一种遗传方法， 为此目的，使用甲醇利用酵母巴斯德毕赤酵母作为 模型 选择这种酵母是因为过氧化物酶体绝对是 在甲醇上生长所需的，一种容易观察到的表型， 因为传统的和分子遗传学的方法 生物体已完全发育。 全面收集 将分离巴斯德毕赤酵母的过氧化物酶体缺陷（PER）突变体， 进行遗传、生物化学和细胞生物学研究， 阐明其主要缺陷的分子基础。 符合 然后利用突变体克隆受影响的特定基因， 将测定和分析每个PER基因DNA序列。 的 从每个基因沿着推导的氨基酸序列与生物化学 分析每个基因突变的影响有助于阐明 每种蛋白质在过氧化物酶体功能中作用， 过氧化物酶体运作的全貌。 该计划的第二个长期目标是利用获得的知识 从这项研究中了解人类疾病状态， 具体来说是齐薇格的受害者 预计一些酵母 所鉴定的过氧化物酶体基因将是 在齐薇格病患者中受到影响。 如果是这样，从酵母菌中获得的信息 PER基因及其产物可用于鉴定和分离 人类同源物 这些人类PER基因将是有价值的， 诊断探针，并最终可用于基因治疗。