QUANTITATIVE TRAIT LOCI ANALYSIS OF OXIDATIVE STRESS MARKERS

这项提案的长期目标是确定影响基因 小鼠衰老通过使用数量性状基因座（QTL）分析。 为了做到这一点，与氧化应激相关的几种分子标记物 将在150岁时进行横断面研究， 使用C57 B1/6 DBA/2（BXD）重组近交系， (RI)品系和BXD F2小鼠;这些标志物的定量差异 菌株和个体间的基因型差异将与基因分型相关 数据在本次重新提交中，对总体研究设计进行了重大修订 初步审查提出的宝贵建议， 这个子项目的分析方法。我们扩大了小组 氧化应激标志物，包括那些影响3个主要 包括大脑在内的3个器官中的大分子类别， 在小鼠中随着年龄的增长而发生显著变化。具体地说， 将通过以下方法评估心脏组织中DNA的氧化损伤： 通过PCR定量线粒体DNA缺失和测量8个 羟基-2 '-脱氧鸟苷。抗氧化剂的蛋白质水平 含铜锌超氧化物歧化酶（CuZn-SOD）和 含锰超氧化物歧化酶（Mn-SOD）将在 肝脏免疫印迹法。还将评估肝脏中的脂质过氧化反应 硫代巴比妥酸检测小脑中的蛋白质氧化 以蛋白质羰基含量定量。这些标志物的定量 氧化应激将与多态性遗传 标记，以确定染色体区域（QTL）影响这些年龄- 相关现象。

The long-term goal of this proposal is to identify genes influencing murine aging through the use of quantitative trait loci (QTL) analysis. To do so, several molecular markers associated with oxidative stress will be measured across the lifespan in a cross-sectional study at 150, 450, and 750 days of age using C57Bl/6 DBA/2 (BXD) recombinant inbred (RI) strains and BXD F2 mice; quantitative differences in these markers among strains and individuals will then be correlated with genotyping data. In this resubmission, major revisions in the overall study design and valuable suggestions by the initial reviews have led to a broadening of the analytic approach for this subproject. We have expanded the panel of oxidative stress markers to include those which affect 3 major classes of macromolecules in 3 organs, including brain, and which have been shown to change significantly with age in mice. Specifically, oxidative damage to DNA in cardiac tissue will be assessed by quantitation of mitochondrial DNA deletions by PCR and measurement of 8 hydroxyl-2'-deoxyguanosine by HPLC. Protein levels of the antioxidant enzymes copper-zinc containing superoxide dismutase (CuZn-SOD) and manganese containing superoxide dismutase (Mn-SOD) will be measured in liver by Western blot. Lipid peroxidation will also be assessed in liver with the thiobarbituric acid assay. Protein oxidation in cerebellum will be quantified as protein carbonyl content. Quantitation of these markers of oxidative stress will then be correlated with polymorphic genetic markers to identify chromosomal regions (QTL) influencing these age- related phenomena.