Novel peroxisomal genes in the yeast Pichia pastoris

毕赤酵母中的新型过氧化物酶体基因

• 批准号: 6760790
• 负责人: James Michael Cregg
• 金额: $ 14.8万
• 依托单位: KECK GRADUATE INST OF APPLIED LIFE SCIS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-07-01 至 2006-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6760790
• 关键词: amines; cell component structure /function; cell proliferation; electron microscopy; fungal genetics; gene deletion mutation; gene expression; gene expression profiling; gene induction /repression; genetic mapping; genetic transcription; methanol; microarray technology; microorganism culture; microorganism growth; molecular assembly /self assembly; mutant; nucleic acid chemical synthesis; nucleic acid sequence; oleate; oligonucleotides; open reading frames; peroxisome; protein localization; yeasts

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.

描述（由申请人提供）：过氧化物酶体是普遍存在的真核细胞器，参与多种重要的代谢途径。在人类中，过氧化物酶体对于某些脂质的代谢是必不可少的，并且其生物发生的缺陷是导致称为过氧化物酶体生物发生障碍（PBD）的致死性遗传障碍家族的原因。该计划的主要长期目标是在分子水平上了解控制过氧化物酶体生物发生的机制。一个组合的分子遗传和生化攻击已经开始使用酵母毕赤酵母作为模型系统。这种酵母的独特之处在于，过氧化物酶体在大小和数量上被大量特异性地诱导，以响应两种碳源，甲醇或油酸中的任一种。首先，通过在巴斯德毕赤酵母和其它酵母中分离pex突变体，已经鉴定了25种不同的PEX基因，并描述了它们的产物（过氧化物酶或PEX蛋白）。很可能许多其他基因参与过氧化物酶体生物发生，但不能通过遗传方法鉴定。 我们建议利用最近获得的巴斯德毕赤酵母基因组序列，并测试通过基因分析方法鉴定新PEX基因的可行性。在本申请中，我们建议构建含有选定的巴斯德毕赤酵母基因子集的微阵列，并利用该阵列来检查响应甲醇和油酸而发生的转录变化。特别是，我们将专注于生理（恒化培养）和遗传（某些突变体背景）的方法，以进一步提高甲醇和油酸诱导的转录反应在巴斯德毕赤酵母的发展。响应于这些碳源之一或两者而被强烈诱导的基因将被鉴定为PEX基因候选基因。将构建缺失各菌株的巴斯德毕赤酵母菌株，并从生化和形态学上检查过氧化物酶体功能，以鉴定新的PEX基因并确定其在过氧化物酶体生物发生中的作用。