Systematic analysis of age-dependently expressed genes in mouse tissues

1.小鼠组织中年龄依赖性表达基因的鉴定我们使用荧光差异显示pcr技术（FDD-PCR）分析小鼠组织中年龄依赖性表达的基因。在小鼠肾脏中发现3个已知基因（纤维连接蛋白、可溶性鸟苷环化酶α1亚基、胞质醛脱氢酶）和3个未知基因，在小鼠脑中发现4个已知基因（髓磷脂蛋白脂蛋白、转铁蛋白、蛋白酪氨酸磷酸酶、g底物）。通过实时荧光定量PCR分析证实了这些基因的年龄依赖性表达。基于FDD-PCR筛选小鼠肾脏中年龄依赖性表达基因获得的EST序列，我们发现了一个编码新蛋白（M-LP）的小鼠基因。M-LP的序列同源性与过氧化物酶体Mpvl7蛋白非常相似。，参与早发性肾小球硬化和PMP22发展的膜蛋白。我们通过双色共聚焦分析证实了M-LP的过氧化物酶体定位，并证明了第一个跨膜片段和下一个环区nh2末端的一半（氨基酸残基15-55）对于过氧化物酶体靶向是必要和充分的。差异渗透实验的结果表明，M-LP由四个跨膜片段组成(TMS I，氨基酸残基15-34；TMS2, 5 1-67; TMS3, 92-110；TMS4, 151-168)和三个中间环，其中n端和c端区域暴露在细胞质中。为了阐明M-LP的功能，我们检测了COS-7细胞中参与活性氧（ROS）代谢的几种酶的活性，发现转染M-LP可显著提高超氧化物歧化酶（SOD）的活性。实时荧光定量PCR结果显示，转染N4-LP后的COS-7细胞锰SOD(50D2)mRNA水平升高。这些结果表明M-LP参与ROS代谢。

1.Identification of age-dependently expressed genes in mouse tissuesWe used a fluorescence differential display-PCR technique(FDD-PCR) to analyze the genes that were age-dependently expressed in mouse tissues.Three known genes(fibronectin, soluble guanylyl cyclase α1 subunit, cytosolic aldehyde dehydrogenase)and 3 unknown genes were found in mouse kidney, and 4 known genes(myelin proteolipid protein, transferrin, protein tyrosine phosphatase, G-substrate)in mouse brain. Age-dependent expression of these genes was confirmed by quantitative real-time PCR analysis.2.A novel peroxisomal membrane protein M-LPWe have identified a mouse gene encoding a novel protein (M-LP), based on an EST sequence obtained by FDD-PCR screening of age-dependently expressed genes in mouse kidney.M-LP has sequence homologies very similar to the Mpvl7 protein, a peroxisomal., membrane protein involved in the development of early-onset glomerulosclerosis and PMP22.We confirmed the peroxisomal localization of M-LP by performing dual color confocal analysis and demonstarted that the first transmembrane segment and NH2-terminal half of the following loop region(amino acid residues 15-55) were necessary and sufficient for peroxisomal targeting..The results of the differential permeabilization experiments revealed that M-LP consists of four transmembrane segments(TMS I, amino acid residues 15-34 ; TMS2, 5 1-67 ; TMS3, 92-110 ; TMS4, 151-168)and three intervening loops, with the N-and C-terminal regions being exposed to the cytosol.In order to elucidate the function of M-LP, we examined the activities of several enzymes involved in reactive oxygen species(ROS)metabolism in COS-7 cellsand found that transfection with M-LP increased the superoxide dismutase(SOD)activity significantly.Quantitative real-time PCR analysis revealed that the manganese SOD(50D2)mRNA level of COS-7 cells transfected with N4-LP was elevated.These results indicate that M-LP participates in ROS metabolism.