報告書番号

U00009

タイトル（和文）

酵母細胞の遺伝子発現におよぼす商用周波磁界の影響 --二次元電気泳動、分子インデックスおよびDNAマイクロアレイ法による、強磁界曝露下での遺伝子発現のゲノムスケール分析--

タイトル（英文）

Effect of Strong Power Frequency Magnetic Field on Gene Expression in Yeast - genome scale analysis of gene expression by two-dimensional electrophoresis, molecular indexing and DNA microarray -

概要 （図表や脚注は「報告書全文」に掲載しております）

商用周波磁界の遺伝子発現に与える影響について真核細胞のモデルである酵母を用い検討した。磁界曝露には、50Hz、10、150、300mTrmsの垂直磁界を用い、静地で嫌気的に培養し、24時間曝露した。陽性対照として、通気培養、熱刺激（40度）および最小培地（栄養源不足）での培養を行った。遺伝子発現は、二次元電気泳動でタンパク質合成を、分子インデックスおよびDNAマイクロアレイでmRNA合成を広範に検討した。その結果、陽性対照ではタンパク質およびmRNAの合成において顕著な変化が確認できたが、磁界曝露においては、いずれの磁界強度においても一貫性のある顕著な変化は見られなかった。このことから、300mT以下の商用周波磁界が、真核細胞における基本的な遺伝子発現機構に作用しないことが示された。

概要 （英文）

Although we have investigated biological effect of power frequency magnetic field (MF) by using bacterial (prokaryotic) cells as simple models, any effects on gene expression, mutagenesis and co-mutagenesis have not been found. If these biological effects in eukaryotic cells exist under the MF exposure, any change in genome-wide gene expression would be detected by exposure to strong MF. In this study, we used stronger MF (10-300mT; million times larger than that which was reported health effect in some epidemiological studies), and investigated the effect of the MF on gene expression in yeast, Saccharomyces cerevisiae , by genome-wide screening method, such as two-dimensional electrophoresis (2-D) for protein synthesis, and molecular indexing (MI) and DNA microarray for RNA synthesis. The yeast cell is widely used as a model eukaryotic cell, since the cell process and the function such as cell cycle regulation, gene expression control, chromosome replication, cell division, sexual reproduction (sporulation), and stress response are very similar to other higher animals including human. The cell also has many gene homologs for human cancer related genes, such as p53, ATM and RAS . Firstly, protein synthesis was estimated by 2-D. Approximately 1,000 proteins of yeast were separated. Stress responsive proteins, such as HSP60, SSA1, SSA2, SSB1, SSC2, were detected. In each positive control, a change in the protein synthesis levels for some stress proteins, such as HSP60 and SSA1 (in heat shock), and other unidentified proteins (in heat shock, aerobic condition, and minimal medium) was observed. No significant change was detected in the 2-D gel of the yeast under exposure to the 10, 150 and 300mT MF for 24h. Secondly, mRNA synthesis was estimated by MI.This method in based on using two class IIS restriction enzymes (Fok I, Bsm AI), 64 adaptors for all possible overhangs and PCR amplified by three anchored oligo dT primer. This method can classify all expressed genes to 384 classes, and the redundancy is minimized. Ideally, The 70% of genes will be displayed. Electropherogram of each class for MF exposed (10mT, 24h) yeast cells are compared with that for control cells (non-exposed). Some positive results were obtained, but the results could not be reproduced. This method is one of powerful methods for seeking gene expression difference, but the throughput is not so high. Finally, mRNA synthesis was estimated by using DNA microarray. The method is the most effective to seek change in gene expression, since this method can compare 95% of total genes. In each positive control, above 50 genes were detected with a change in mRNA synthesis level. Neither significant nor consistent change was detected on the microarray of the cells between control and the MF exposure (10, 150 and 300mT) for 24h. These results suggest that the strong MF could not affect a fundamental mechanism of mRNA synthesis or protein synthesis in the model eukaryotic cell.

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