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利用荧光差异显示-聚合酶链式反应技术对小鼠组织中的年龄依赖性表达基因进行了分析，在小鼠肾脏中发现了3个已知基因(纤维连接蛋白、可溶性鸟苷酸环化酶α1亚基、胞浆乙醛脱氢酶)和3个未知基因，在小鼠脑中发现了4个已知基因(髓鞘蛋白脂蛋白、转铁蛋白、蛋白酪氨酸磷酸酶、G底物)。2.一种新的过氧体膜蛋白M-LP我们根据FDD-PCR筛选小鼠肾脏中与年龄相关的表达基因的EST序列，鉴定了一个编码新蛋白(M-LP)的小鼠基因。M-LP与过氧体Mpvl7蛋白具有非常相似的序列同源性。膜蛋白参与早发性肾小球硬化和PMP22的发生发展。我们通过双色共聚焦分析证实了M-LP的过氧体定位，并证明了第一跨膜段和随后环区的NH2末端半部分(氨基酸残基15-55)是过氧体靶向的必要条件和充分条件。为了阐明M-LP在COS-7细胞中的功能，我们检测了M-LP在COS-7细胞中参与ROS代谢的几种酶的活性，发现M-LP显著提高了超氧化物歧化酶(SOD)的活性。实时荧光定量PCR分析表明，转导N4-LP的COS-7细胞中锰超氧化物歧化酶(50D2)的mRNA水平显著升高，提示M-LP参与了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.