ISOLATION OF GENES INVOLVED IN CYTOCHROME BIOGENESIS

细胞色素生物发生相关基因的分离

• 批准号: 2172454
• 负责人: BETH W DREYFUSS
• 金额: $ 2.37万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 1995
• 资助国家: 美国
• 起止时间: 1995-12-30 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/2172454
• 关键词: ISOLATION; GENES; INVOLVED; CYTOCHROME; BIOGENESIS

The overall objective of the research in this proposal is to investigate the biochemical steps that occur during the post-translational maturation of protein-cofactor complexes. The specific goal includes the cloning and characterization of at least one gene that is required for attachment of heme during the assembly of cyt f and cyt c6 in chloroplasts, and the determination of the function of the gene product. Cytochromes f and c6 are members of a highly conserved family of c-type cytochromes that are ubiquitous in all energy transducing membranes. The experiments will exploit Chlamydomonas, since it is well-suited for approaches involving both genetics and molecular biology. The work involves the generation and identification of tagged nuclear mutants that are defective in cyt c6 and cyt f accumulation, the demonstration that these are indeed heme attachment mutants and that the locus of interest is tagged, followed by the cloning of the corresponding gene on the basis of the tag. "Tagged" mutants will be generated by random integration of the argininosuccinate lyase (ASL) gene into the nuclear genome by transformation of a recipient arg-minus strain. Mutants will be identified among the arg-plus transformants on a fluorescence video imaging system based on their loss of photosynthetic capacity and by biochemical assays for failure to accumulate holocyt c6 and holocyt f. Complementation of previously- characterized nuclear heme attachment mutants using an indexed genomic cosmid library or chloroplast mutants using cloned chloroplast DNA is proposed as a parallel strategy. The project has revelance to human health because the same principles apply to respirator cytochromes, and thus will enhance our understanding and hence treatment of mitochondrial myopathies.

该提案中研究的总体目的是调查 翻译后成熟期间发生的生化步骤 蛋白质 - 配合物复合物。具体目标包括克隆和 至少一个基因的表征是附着的 血红素在叶绿体中的Cyt F和Cyt C6组装过程中，以及 确定基因产物功能。细胞色素F和C6 是高度保守的C型细胞色素家族的成员 无处不在的所有能量转导膜。实验会 利用衣原体，因为它非常适合涉及的方法 遗传学和分子生物学。这项工作涉及一代和 鉴定在Cyt C6和 Cyt F积累，这些证明确实是血红素 附着突变体，并标记了感兴趣的座位，其次是 基因基于标签的克隆。 “标记” 突变体将通过随机整合精氨酸核酸盐而产生 通过转化，裂解基因（ASL）基因进入核基因组 arg-minus菌株。突变体将在Arg-Plus中识别 基于荧光视频成像系统的转化子根据其损失 光合作用能力和通过生化测定未能 积累Holocyt C6和Holocyt f。以前的互补 使用索引的基因组表征核血红素附着突变体 使用克隆叶绿体DNA的宇宙库或叶绿体突变体为 提议作为平行策略。该项目对人类有启示 健康，因为相同的原理适用于呼吸器细胞色素，并且 因此，将增强我们的理解并因此对线粒体的治疗 肌病。