Novel peroxisomal genes in the yeast Pichia pastoris

描述(申请人提供)：过氧化物体是广泛存在的真核细胞器，参与多种重要的代谢途径。在人类中，过氧酶体是某些脂质代谢所必需的，其生物发生的缺陷导致了一系列称为过氧酶体生物发生障碍(PBDS)的致命遗传疾病。这项计划的主要长期目标是在分子水平上了解控制过氧化物体生物发生的机制。以巴斯德毕赤酵母为模型系统，发起了一场分子遗传和生化联合攻击。这种酵母的独特之处在于，过氧化体在大小和数量上被大量和特异地诱导，以响应两个碳源之一，甲醇或油酸。主要通过分离巴斯德毕赤酵母和其他酵母中的PEX突变体，已经鉴定了25个不同的PEX基因，并描述了它们的产物(过氧化物素或PEX蛋白)。很可能还有许多其他基因参与了过氧化酶体的生物发生，但不能用遗传学的方法来识别。 我们建议利用最近获得的巴斯德毕赤酵母基因组序列，并测试通过基因图谱方法识别新的PEX基因的可行性。在这项应用中，我们建议构建包含选定的巴斯德毕赤酵母基因子集的微阵列，并利用这些阵列来检测发生在对甲醇和油酸的反应中的转录变化。特别是，我们将专注于生理(恒化器培养)和遗传(某些突变背景)方法的发展，以进一步增强甲醇和油酸诱导的巴斯德毕赤酵母的转录反应。对这两种碳源中的一种或两种反应强烈诱导的基因将被确定为PEX基因候选。将构建每个菌株缺失的巴斯德毕赤酵母菌株，并对其进行生化和形态检查，以确定新的PEX基因，并确定它们在过氧酶体生物发生中的作用。

DESCRIPTION (provided by applicant): Peroxisomes are ubiquitous eukaryotic organelles involved in a variety of important metabolic pathways. In humans, peroxisomes are essential for the metabolism of certain lipids, and defects in their biogenesis are responsible for a family of lethal genetic disorders termed peroxisome biogenesis disorders (PBDs). The primary long-term goal of this program is to understand, at the molecular level, the mechanisms controlling peroxisomal biogenesis. A combined molecular genetic and biochemical attack has been initiated using the yeast Pichia pastoris as the model system. This yeast is unique in that peroxisomes are massively and specifically induced in size and number in response to either of two carbon sources, methanol or oleic acid. Primarily through the isolation ofpex mutants in P. pastoris and other yeasts, 25 different PEX genes have been identified and their products, (peroxins or PEX proteins) have been described. It is likely that many other genes are involved in peroxisome biogenesis but cannot be identified by the genetic approach. We propose to take advantage of the recent availability of the P. pastoris genomic sequence and test the feasibility of identifying new PEX genes via a gene profiling approach. In this application, we propose to construct microarrays containing a selected subset of P. pastoris genes and utilize the arrays to examine changes in transcription that occur in response to methanol and oleic acid. In particular, we will focus on the development of physiological (chemostat culturing) and genetic (certain mutant backgrounds) methods to further enhance the methanol- and oleate-induced transcriptional responses in P. pastoris. Genes that are strongly induced in response to either or both of these carbon sources will be identified as PEXgene candidates. P. pastoris strains deleted for each strain will be constructed and examined biochemically and morphologically for peroxisome function to identify new PEX genes and to define their role in peroxisome biogenesis.