To create the unique properties of a certain cellular membrane, both the composition and the metabolism of membrane phospholipids are key factors. Phospholipase A 2 (PLA 2 ), with hydrolytic enzyme activities at the sn-2 position in glycerophospholipids, plays critical roles in maintaining the phospholipid composition as well as producing bioactive lipid mediators. In this study we examined the contribution of a Ca 2؉ -independent group IVC PLA 2 isozyme (cPLA 2 ␥), a paralogue of cytosolic PLA 2 ␣ (cPLA 2 ␣), to phospholipid remodeling. The enzyme was localized in the endoplasmic reticulum and Golgi apparatus, as seen using green fluorescence fusion proteins. Electrospray ionization mass spectrometric analysis of membrane extracts revealed that overexpression of cPLA 2 ␥ increased the proportion of polyunsaturated fatty acids in phosphatidylethanolamine, suggesting that the enzyme modulates the phospholipid composition. We also found that H 2 O 2 and other hydroperoxides induced arachidonic acid release in cPLA 2 ␥-transfected human embryonic kidney 293 cells, possibly through the tyrosine phosphorylation pathway. Thus, we propose that cPLA 2 ␥ is constitutively expressed in the endoplasmic reticulum and plays important roles in remodeling and maintaining membrane phospholipids under various conditions, including oxidative stress.

Restriction landmark genome scanning (RLGS) is a 2-DE of genomic DNA, which visualizes thousands of loci. In a conventional RLGS method for methylation analysis, we have used a methylation sensitive restriction enzyme, NotI as a landmark. However, it was unable to discriminate methylation polymorphism from sequence polymorphism. Here, we report an improved RLGS method to detect methylated sites directly. We employed isoschizomers, MspI and HpaII, that recognize the same sequence (CCGG) but have different methylation sensitivity. We carried out the RLGS analysis of Arabidopsis thaliana ecotype Columbia, and obtained a pair of spot patterns with MspI and HpaII. We detected 22 spots in both patterns. In comparison of them, 18% of the spots were polymorphic, which indicated the methylation of C(5m)CGG sites. Further analyses revealed an additional methylated site of NotI. Moreover, 52 and 54 restriction enzyme sites were also analyzed in two other ecotypes, Wassilewskija and Landsberg erecta, respectively. Consequently, 15% of the 52 common sites showed methylation polymorphism among the three ecotypes. The restriction sites analyzed in this study were located in or near genes, and contribute new data about the correlation between methylation status and gene expression. Therefore, this result strongly indicates that the improved RLGS method is readily applicable to practical analyses of methylation dynamics, and provides clues to the relationship between methylation and gene expression.

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