| Research Abstract |
1.Identification of age-dependently expressed genes in mouse tissues
We 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-LP
We 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.
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