Title 酵母Saccharomyces cerevisiaeにおける圧力感受性機構の解明 ( 本文(Fulltext) ) Author(s) 野村, 一樹 Report No.(Doctoral Degree) 博士(農学) 甲第645号 Issue Date 2015-03-31 Type 博士論文 Version ETD URL http://repository.lib.gifu-u.ac.jp/handle/123456789/51019 ※この資料の著作権は、各資料の著者・学協会・出版社等に帰属します。 㓝ẕ Saccharomyces cerevisiae ࠾ࡅࡿᅽຊឤཷᛶᶵᵓࡢゎ᫂ 2014 ᖺ ᒱ㜧ᏛᏛ㝔㐃ྜ㎰Ꮫ◊✲⛉ ⏕≀㈨※⛉Ꮫ 㸦ᒱ㜧Ꮫ㸧 㔝 ᮧ ୍ ᶞ 㓝ẕ Saccharomyces cerevisiae ࠾ࡅࡿᅽຊឤཷᛶᶵᵓࡢゎ᫂ 㔝 ᮧ ୍ ᶞ ┠ḟ ➨ 1 ❶ ᗎㄽ...................................................................................................... 1 ➨ 1 ⠇ 㟼Ỉᅽ ............................................................................................... 1 ➨ 1 㡯 ᅽຊࡢ≉ᛶ .................................................................................... 2 ➨ 2 㡯 㧗ᅽࡼࡿᚤ⏕≀ࡢάᛶ ........................................................ 3 ➨ 3 㡯 㧗ᅽ◊✲ࡢṔྐ㸫ẼᅽࡢⓎぢࡽ㧗ᅽ◊✲ࡢⓎᒎ㸫 ................ 5 ➨ 4 㡯 㧗ᅽ◊✲ࡢṔྐ㸫㣗ရࡢ㧗ᅽຍᕤࡢᛂ⏝㸫 ............................... 8 ➨ 2 ⠇ 㓝ẕ ................................................................................................ 12 ➨ 1 㡯 ࣔࢹࣝ⏕≀ࡋ࡚ࡢ㓝ẕ ............................................................. 13 ➨ 2 㡯 Ⓨ㓝㣗ရࡋ࡚ࡢ㓝ẕ ................................................................ 15 ➨ 3 ⠇ 㧗ᅽ㣗ရຍᕤᢏ⾡ ........................................................................... 16 ➨ 1 㡯 㧗ᅽຍᕤ㣗ရࡢ㛤Ⓨၥ㢟Ⅼ ...................................................... 16 ➨ 2 㡯 Pressure Regulated Fermentation ............................................ 17 ➨ 3 㡯 ᅽຊឤཷᛶ㓝ẕࡢసฟ ................................................................ 18 ➨ 4 ⠇ ◊✲┠ⓗ ......................................................................................... 19 ➨ 2 ❶ DNA ࣐ࢡࣟࣞࡼࡿ⥙⨶ⓗ㑇ఏᏊⓎ⌧ゎᯒ .......................... 20 ➨ 1 ⠇ ⥴ゝ ................................................................................................ 20 ➨ 1 㡯 DNA ࣐ࢡࣟࣞゎᯒ ........................................................... 20 ➨ 2 㡯 DNA ࣐ࢡࣟࣞゎᯒࡢᛂ⏝............................................. 21 ➨ 3 㡯 DNA ࣐ࢡࣟࣞゎᯒ࠾ࡅࡿࢡࣟࢫࣁࣈࣜࢲࢮ࣮ࢩࣙࣥ ... 22 ➨ 4 㡯 ᐇ㦂┠ⓗ ...................................................................................... 23 ➨ 2 ⠇ ᐇ㦂ᮦᩱ࠾ࡼࡧᐇ㦂᪉ἲ ................................................................ 24 ➨ 1 㡯 ⏝⳦ᰴ ...................................................................................... 24 ➨ 2 㡯 ᇵ㣴᮲௳ ...................................................................................... 24 ➨ 3 㡯 RNA ᢳฟ᪉ἲ ............................................................................. 24 ➨ 4 㡯 DNA ࣐ࢡࣟࣞゎᯒ᪉ἲ .................................................... 25 ➨ 5 㡯 㑇ఏᏊⓎ⌧ࡢศ㢮ゎᯒ᪉ἲ ......................................................... 26 ➨ 6 㡯 quantitative PCR ᪉ἲ ............................................................... 26 ➨ 3 ⠇ ⤖ᯝ ................................................................................................ 28 ➨ 1 㡯 㑇ఏᏊⓎࣉࣟࣇࣝࡢᴫせ ...................................................... 28 ➨ 2 㡯 ࢵࣉࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊⓎ⌧ࡢゎᯒ ....................................... 28 ➨ 3 㡯 ࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊⓎ⌧ࡢゎᯒ ....................................... 30 ➨ 4 㡯 quantitative PCR ࡼࡿ㑇ఏᏊⓎ⌧ゎᯒࡢホ౯ ........................ 31 ➨ 4 ⠇ ⪃ᐹ ................................................................................................ 32 ➨ 3 ❶ ࣑ࢺࢥࣥࢻࣜᶵ⬟ࡢゎᯒ ................................................................ 35 ➨ 1 ⠇ ⥴ゝ ................................................................................................ 35 ➨ 1 㡯 㓝ẕ࣑ࢺࢥࣥࢻࣜ ................................................................ 35 ➨ 2 㡯 ᐇ㦂┠ⓗ ...................................................................................... 36 ➨ 2 ⠇ ᐇ㦂ᮦᩱ࠾ࡼࡧᐇ㦂᪉ἲ ................................................................ 37 ➨ 1 㡯 ⏝⳦ᰴ ...................................................................................... 37 ➨ 2 㡯 ᇵ㣴᮲௳ ...................................................................................... 37 ➨ 3 㡯 ಸయᰴࡢసฟ ........................................................................... 37 ➨ 4 㡯 ྾ᶵ⬟ࡢゎᯒ᪉ἲ .................................................................... 38 ➨ 5 㡯 ࣑ࢺࢥࣥࢻࣜ DNA Ḟኻࡢゎᯒ᪉ἲ ........................................ 38 ➨ 6 㡯 㧗ᅽฎ⌮᪉ἲ ............................................................................... 39 ➨ 3 ⠇ ⤖ᯝ ................................................................................................ 40 ➨ 1 㡯 ྾ᶵ⬟ࡢゎᯒ ........................................................................... 40 ➨ 2 㡯 ࣑ࢺࢥࣥࢻࣜ DNA ࡢḞኻ ...................................................... 40 ➨ 3 㡯 ಸయᰴࡢసฟ ........................................................................... 41 ➨ 4 㡯 ᅽຊάᛶᣲືࡢゎᯒ ............................................................. 42 ➨ 5 㡯 㔝⏕ᆺ࣑ࢺࢥࣥࢻࣜࡼࡿḞኻ㑇ఏᏊࡢ⿵ ......................... 42 ➨ 4 ⠇ ⪃ᐹ ................................................................................................ 44 ➨ 4 ❶ ࣓ࢱ࣑࣎ࣟࢡࢫࡼࡿᅽຊឤཷᛶᶵᵓࡢゎᯒ ................................... 47 ➨ 1 ⠇ ⥴ゝ ................................................................................................ 47 ➨ 1 㡯 ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒ .................................................................... 47 ➨ 2 㡯 ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒࡢᛂ⏝ ...................................................... 48 ➨ 3 㡯 ࣝࢠࢽࣥ .................................................................................. 48 ➨ 4 㡯 ᐇ㦂┠ⓗ ...................................................................................... 49 ➨ 2 ⠇ ᐇ㦂ᮦᩱ࠾ࡼࡧᐇ㦂᪉ἲ ................................................................ 50 ➨ 1 㡯 ⏝⳦ᰴ ...................................................................................... 50 ➨ 2 㡯 ᇵ㣴᮲௳ ...................................................................................... 50 ➨ 3 㡯 ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒ᪉ἲ ............................................................. 50 ➨ 4 㡯 ௦ㅰ⤒㊰ࡢゎᯒ᪉ἲ .................................................................... 51 ➨ 5 㡯 㧗ᅽฎ⌮᪉ἲ ............................................................................... 52 ➨ 3 ⠇ ⤖ᯝ ................................................................................................ 53 ➨ 1 㡯 ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒ .................................................................... 53 ➨ 2 㡯 ࣝࢠࢽࣥ௦ㅰゎᯒ⤒㊰㛵ࡍࡿ㑇ఏᏊࡢゎᯒ ......................... 53 ➨ 3 㡯 ࣝࢠࢽࣥࡢᅽຊάᛶࡢᐤ ........................................... 54 ➨ 4 ⠇ ⪃ᐹ ................................................................................................ 55 ➨ 5 ❶ ⤖ㄽ.................................................................................................... 58 ➨ 6 ❶ ㅰ㎡.................................................................................................... 64 ➨ 7 ❶ ཧ⪃ᩥ⊩ ............................................................................................ 66 ➨ 8 ❶ ᅗ⾲.................................................................................................... 80 ➨ 1 ❶ ᗎㄽ ➨ 1 ⠇ 㟼Ỉᅽ ᅽຊ (pressure) ࡣࠊ༢㠃✚࠶ࡓࡾస⏝ࡍࡿຊᐃ⩏ࡉࢀࡿ⇕ຊᏛⓗࣃ ࣓࣮ࣛࢱࡢ୍✀࡛࠶ࡿࠋᮏ◊✲࡛⏝࠸ࡿ㟼Ỉᅽ (hydrostatic pressure) ࡣࠊỈ➼ ࡢᾮయࢆ፹యࡋࡓᅽຊࡢࡇ࡛࠶ࡿࠋ௨ୗࠊ㟼Ỉᅽࡣᅽຊグ㍕ࡍࡿࠋ᭱ࡶ㌟ ㏆Ꮡᅾࡍࡿᅽຊࡢࡋ࡚Ẽᅽࡀ࠶ࡿࠋᆅ⾲ࡢẼᅽࡣ࠾ࡼࡑ 1 Ẽᅽ࡛࠶ ࡾࠊྂࡃࡣẼࢆពࡍࡿ atmosphere ࡽ 1 atm ࡶ⾲ࡉࢀࡓࠋ⌧ᅾ࡛ࡣᅜ㝿༢ ⣔ࡼࡾ 1 Ẽᅽ=1,013 hPaҸ0.1 MPa ⾲♧ࡍࡿࠋ1 Pa (ࣃࢫ࢝ࣝ)ࡣࠊ1 N/m2 ᐃ⩏ࡉࢀ࡚࠸ࡿࠋẼᅽࡣẼࡢ㔜ࡉࡶ⪃࠼ࡽࢀࠊ㧗ᗘࡀ㧗ࡃ࡞ࡿࠊẼ ᅽࡣపࡃ࡞ࡿࠋࡲࡓࠊỈ୰࡛ࡣỈ῝ࡀ 10 m ῝ࡃ࡞ࡿẖ 1 ẼᅽҸ0.1 MPa ࡎࡘ Ỉᅽࡀ㧗ࡃ࡞ࡾࠊᆅ⌫ୖ࡛ 1 ␒῝࠸࣐ࣜࢼᾏ⁁ࡣỈ῝ 10,911 mࠊ࠾ࡼࡑ 1,100 ẼᅽҸ110 MPa ࡢ㧗ᅽࡢୡ⏺࡛࠶ࡿࠋ ➨ 1 㡯 ᅽຊࡢ≉ᛶ ᅽຊࡣࠊ ᗘྠᵝࢠࣈࢬ࢚ࢿࣝࢠ࣮ࢆኚࡉࡏࡿࣃ࣓࣮ࣛࢱ࡛࠶ࡾࠊ࠶ࡿ ⣔ࡢ≧ែࢆኚࡉࡏࡿࡓࡵ⾜࡛ࡁࡿ࢚ࢿࣝࢠ࣮࡛࠶ࡿࠋ࠼ࡿ࡞ࡽࡤࠊ㣗ရ ࡢຍᕤࡀࢃࡾ᫆࠸ࠋ㣗ရࡣຍ⇕ࡍࡿࡇࡼࡾࠊ㣗ရ୰ࡢᵝࠎ࡞ศᏊ㐠ືࡀά Ⓨ࡞ࡾࠊỈ➼ࡢⓎᡂศࡀẼࡋ࡚ᾘኻࡋࠊศᏊྠኈࡢ⾪✺ᅇᩘࡀቑ࠼࡚ศᏊ 㛫ࡢᏛᛂࡀಁ㐍ࡉࢀࡿ(ᒣᮏ, ᑠ㛵, 2009)ࠋࡑࡢ⤖ᯝࠊศᏊ୰ࡢᐁ⬟ᇶࡀࡢ ᐁ⬟ᇶᛂࡋ࡚᪂ࡓ࡞ຍ⇕⏕ᡂ≀ࢆ⏕ࡌࡓࡾࠊඹ᭷⤖ྜࡀ㛤ࡋ࡚᭷⏝ᡂศ ࡀኻࢃࢀࡓࡾࡍࡿࠋ୍᪉ࠊ㧗ᅽࡼࡿຍᕤᢏ⾡ࡣࠊ✚ᴟⓗ࡞ຍ⇕ࢆకࢃ࡞࠸ᗈ⩏ ࡢ㠀⇕ⓗฎ⌮࡛࠶ࡿࡓࡵࠊୖグࡢ⇕ฎ⌮࡛ಁ㐍ࡉࢀࡿᏛᛂࡣཎ๎ⓗ㉳ ࡇࡽ࡞࠸ࠋࡍ࡞ࢃࡕࠊ㧗ᅽୗ࠾࠸࡚ࡣඹ᭷⤖ྜࡢ᪂ࡓ࡞⏕ᡂࡸ㛤ࡣ㉳ࡇࡽࡎࠊ 㠀ඹ᭷⤖ྜࡢࡳࡀᙳ㡪ࢆཷࡅࡿ(ᯘ, 1991)ࠋ࠼ࡤࠊࢱࣥࣃࢡ㉁➼ࡢᕧศᏊ࡛ ࡣࠊࡑࡢ❧యᵓ㐀ෆࡢ✵㝽ࢆᇙࡵࡿࡼ࠺ࠊࡑࢀࡲ࡛ศᏊࢆᏳᐃࡋ࡚࠸ࡓศᏊ ෆࡢỈ⣲⤖ྜ➼ࡢ㠀ඹ᭷⤖ྜࡀ㛤ࡍࡿࠋࡑࡋ࡚ศᏊ⮬యࡢయ✚ࢆᑠࡉࡃࡍࡿ ࡼ࠺ࠊศᏊෆࡢ✵㝽ࡀᇙࡵࡽࢀ࡚ඖࡢ❧యᵓ㐀ࡀᔂࢀࠊኚᛶࡀᘬࡁ㉳ࡇࡉࢀࡿ (ᒣᮏ, ᑠ㛵, 2009)ࠋ 㧗ᅽࡣ⇕ྠࡌࡼ࠺ࢱࣥࣃࢡ㉁ࢆኚᛶࡉࡏࡿࡀࠊୖグࡢ ࡼ࠺ࡑࡢኚᛶ࣓࢝ࢽࢬ࣒ࡣ␗࡞ࡿࠋࡇࡢࡇࡣຍ⇕࡛ࡣᘬࡁ㉳ࡇࡏ࡞࠸⌧㇟ ࢆຍᅽࡼࡾᘬࡁ㉳ࡇࡍࡇࢆྍ⬟ࡋࠊࡲࡓࡑࡢ㏫ࡶ㉳ࡇࡾᚓࡿࡇࢆ♧ࡋ ࡚࠸ࡿࠋ 2 ➨ 2 㡯 㧗ᅽࡼࡿᚤ⏕≀ࡢάᛶ ᅽຊࡣࠊ㠀ඹ᭷⤖ྜస⏝ࡋ࡚㠀⇕ⓗࢱࣥࣃࢡ㉁ࢆኚᛶࡉࡏࡿࠋࡑࡢࡓࡵ 㧗ᅽࡣ⏕≀ᑐࡍࡿ≀⌮ⓗࢫࢺࣞࢫ࡛࠶ࡿゝ࠼ࡿࠋࡇࢀࡲ࡛㧗ᅽࡼࡿᚤ ⏕≀ࡢቑṪ㜼ᐖࡸάᛶ㛵ࡋ࡚ከࡃࡢሗ࿌ࡀ࠶ࡿ(ZoBell and Cobet, 1964; Iwahashi et al., 1991; Tamura et al., 1992; Abe and Kato, 1999; Vogel et al., 2005; Kawarai et al., 2006)ࠋ࠼ࡤࠊ࠸ࡃࡘࡶࡢⓎ㓝㣗ရࡢ⏕⏘⏝ࡉࢀࡿฟⱆ㓝ẕ Saccharomyces cervisiae ࡣࠊ ᗘ᮲௳ࡼࡿࡀ 150 MPa ௨ୖࡢ㧗ᅽࡣ⮴Ṛⓗ࡞ᦆ യࢆ࠼ࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Nomura et al., 2014)ࠋࡲࡓࠊ40 MPa ⛬ᗘࡢ㧗 ᅽ᮲௳࡛ࡣࠊ4°C ࡛ࡣ⮴Ṛⓗ࡞ᦆയࢆཷࡅࡿࡀࠊ25°C ࡛ࡣቑṪࡍࡿࡇࡀྍ⬟ ࡛࠶ࡿ(Iwahashi et al., 2003)ࠋHashizume ࡽ(1995)ࡣ 120~300 MPaࠊ-20~50°Cࠊ2~40 min ࡢ㧗ᅽຊ᮲௳࡛㓝ẕࡢάᛶࢆ㏿ᗘㄽⓗゎᯒࡋࡓࠋࡑࡢ⤖ᯝࠊ180 MPa ௨ୗࡢ㧗ᅽຊ⠊ᅖ࠾࠸࡚ࠊ0~40°C ࡢ ᗘ⠊ᅖ࡛ࡣࢇάᛶࡀㄆࡵࡽ ࢀ࡞ࡗࡓࡀࠊ-10°C ௨ୗ࠾ࡼࡧ 50°C ௨ୖࡢ ᗘᇦ࡛ࡣྠᅽຊ࡛ࡁ࡞άᛶ ࡀㄆࡵࡽࢀࡓࠋ≉-20°C ࠾ࡅࡿάᛶຠᯝࡀ㢧ⴭ࡛࠶ࡾࠊప ᇦ࡛ࡢ㧗 ᅽຊຠᯝࡢ᭷ຠᛶࡀ♧ࡉࢀࡓࠋ㧗ᅽ᮲௳ୗ࡛ࡣỈࡣịⅬୗ࡛࠶ࡗ࡚ࡶᾮయࡢ≧ ែ࡛Ꮡᅾࡍࡿࡇࡀྍ⬟࡛࠶ࡿࠋ㧗ᅽ࠾ࡼࡧప ࡢ᮲௳ࡣࡕࡽࡶศᏊ㐠ືࢆ పୗࡉࡏࠊ⣽⬊⭷ࡢὶືᛶࢆపୗࡉࡏࡿࡼ࠺ᶵ⬟ࡍࡿࠋ⣽⬊⭷ࡢὶືᛶࡢపୗ ࡣࠊࣜࣥ⬡㉁➼ࡢ⭷ࢱࣥࣃࢡ㉁ࡢ┦㌿⛣ࡼࡾᘬࡁ㉳ࡇࡉࢀࡿ(Chong et al., 1985)ࠋ 3 ࡑࡢ⤖ᯝࠊ⭷ᵓ㐀ࡀ◚ቯࡉࢀࠊ⣽⬊ࡀάᛶࡍࡿ⪃࠼ࡽࢀ࡚࠸ࡿ(Freitas et al., 2012)ࠋ Escherichia coli ⣽⬊㧗ᅽฎ⌮ࢆࡋ࡚άᛶࢆ☜ㄆࡋࡓᚋࠊࣜࣥ㓟ࣂࢵ ࣇ࣮(PBS)୰࡛ 25°Cࠊ1 week ᇵ㣴ࡍࡿࠊࡑࢀࡽࡢ⣽⬊ࡀᅇࡍࡿ࠸࠺⌧㇟ ࡀሗ࿌ࡉࢀࡓ(Koseki and Yamamoto, 2006)ࠋάᛶࡋࡓ⣽⬊ࡀⅣ⣲※ࡸ❅⣲※ ࡞ࡿᰤ㣴ࡢ࡞࠸ PBS ୰࡛ᅇࡍࡿࡇࡣ⪃࠼㞴࠸ࠋOhshima ࡽ(2013)ࡣࠊ㧗ᅽ ࡼࡾάᛶࡋࡓ⣽⬊ࡀ PBS ୰࡛ᅇࡍࡿ࣓࢝ࢽࢬ࣒ࢆゎᯒࡋࡓࠋᙼࡽࡣࠊ 㧗ᅽࡼࡾぢࡅୖάᛶࡉࢀࡓ⣽⬊⩌࠾࠸࡚ࠊ㧗ᅽࡼࡿᦆയࢆཷࡅ࡚ ࠸ࡿࡀάᛶࡋ࡚࠸࡞࠸⣽⬊ࡀഹᏑᅾࡍࡿࡇࢆ᫂ࡽࡋࡓࠋ㧗ᅽᦆ യ⣽⬊ࡣࠊ4°C ࡛ಖᏑࡋ࡚࠸ࡿሙྜ࠾࠸࡚ࡣᅇࡍࡿࡇࡣ࡞࠸ࠋࡋࡋࠊ 25°C ࡢ ᗘ᮲௳࡛ࡣࠊ㧗ᅽᦆയࡽᅇࡋࠊࡑࡢᚋࠊ࿘ᅖࡢάᛶࡋࡓ⣽⬊ ࢆᰤ㣴※ࡋ࡚㧗ᅽฎ⌮ࡍࡿ๓ࡢ⣽⬊ᩘࡢ 50%⛬ᗘࡲ࡛ቑṪࡍࡿࡇࡀ᫂ࡽ ࡞ࡗࡓࠋࡇࢀࡽࡢሗ࿌ࡣࠊάᛶࡋࡓ⣽⬊࡛࠶ࡗ࡚ࡶ ᗘ᮲௳ࡼࡗ࡚ࡣᅇ ࡋࠊቑṪࡍࡿࡇࢆ♧ࡋ࡚࠾ࡾࠊ㣗ရຍᕤ㧗ᅽฎ⌮ࢆᛂ⏝ࡍࡿሙྜࠊฎ⌮ᚋ ࡢ ᗘ⟶⌮ࡀ㔜せ࡛࠶ࡿࡇࢆ♧ࡋ࡚࠸ࡿࠋ 4 ➨ 3 㡯 㧗ᅽ◊✲ࡢṔྐ㸫ẼᅽࡢⓎぢࡽ㧗ᅽ◊✲ࡢⓎᒎ㸫 ᅽຊ࠸࠺ᴫᛕࡀㄆ▱ࡉࢀࡓࡢࡣ 17 ୡ⣖࡛࠶ࡿࠋኳᩥᏛࡢ∗ࡋ࡚ྡ㧗࠸ Galilei ࡑࡢᘵᏊ Torricelli ࡼࡗ࡚┿✵ࡢᴫᛕࡀⓎぢࡉࢀࡓࡢࡀ 1644 ᖺ࡛࠶ ࡿࠋTorricelli ࡣࠊỈ㖟࡛‶ࡓࡋࡓ࢞ࣛࢫ⟶ࢆỈ㖟ᾎᵴ୰ಽ❧ࡉࡏࡿࠊ࠾ࡼࡑ 76 cm ࡢ㧗ࡉࡲ࡛ࡀỈ㖟࡛ࡑࢀࡼࡾୖࡢ㒊ศࡀ┿✵࡞ࡿࡇࢆ᫂ࡽࡋࡓࠋ ࡇࡢཎ⌮ࢆᛂ⏝ࡋࡓỈ㖟ẼᅽィࢆⓎ᫂ࡋࡓຌ⦼ࡽࠊᅽຊࡢ༢ࠕࢺࣝ; Torrࠖ ࡣ Torriceli ࡢྡࡕ࡞ࢇ࡛࠸ࡿࠋࡑࡋ࡚ࡑࡢ 4 ᖺᚋࡢ 1648 ᖺࠊPascal ࡼࡿ Ẽᅽࡢㄆ㆑ࡼࡗ࡚ᅽຊ࠸࠺ᴫᛕࡀึࡵ࡚⛉Ꮫྐグࡉࢀࡓ(Pascal, 1653)ࠋ Pascal ࡣࠊỈ㖟Ẽᅽィࢆ⏝࠸࡚ࠊᆅ⾲ᩍࡢᒇ᰿ࠊᆅ⾲ࣆ࣭ࣗࢻ࣭ࢻ࣮࣒ ᒣࡢ㡬ୖ࠾࠸࡚Ỉ㖟ᰕࡢ㧗ࡉࡀኚࡍࡿࡇࢆぢฟࡋࠊẼᅽࡢᏑᅾࢆド᫂ ࡋࡓࠋࡲࡓᙼࡣࠊᅽຊ(pressure)ࡀᅽ㏕(press)␗࡞ࡿῶᑡ࡛࠶ࡿࡇࢆ ͆⼚ࡣ ᣦᣳࢇ࡛ࡕࡻࡗᢲࡏࡤࡘࡪࢀࡿࡀ㧗࠸ᅽຊࡢୡ⏺࡛ࡣࡘࡪࢀ࡞࠸͇ㄝ᫂ ࡋ࡚࠸ࡿ(ᯘ, 2008)ࠋẼᅽࢆㄆ㆑ࡋࡓຌ⦼ࡽࠊ᪥࡛ࡣᅽຊ༢ࡢࡇࢆࠕࣃ ࢫ࢝ࣝ; Paࠖࡪࠋ 1905 ᖺࡣࠊ㧗ᅽ◊✲ࡢṔྐṧࡿ❅⣲Ỉ⣲ࡽࣥࣔࢽࡢᏛྜᡂࡀ ᡂຌࡋࡓࠋHaber ࡣᖖ ᖖᅽ࡛ࡣ㐍⾜ࡋ࡞࠸ࡇࡢᏛᛂᑐࡋ࡚ࠊ200-500°Cࠊ 20-100 MPa ࠸࠺㧗 㧗ᅽ᮲௳㕲ࢆయࡋࡓゐ፹ࢆ⏝࠸ࡿࡇࡼࡗ࡚ ࣥࣔࢽྜᡂἲࢆ☜❧ࡋࡓ(㔜ᯇ, 2013)ࠋࡑࡢᚋࠊBosch ࡼࡾࣁ࣮ࣂ࣮࣭࣎ࢵࢩ 5 ࣗἲࡤࢀࡿࣥࣔࢽᏛྜᡂࡀၟᴗࡉࢀࡓࠋᮏἲࡣ᪥࠾࠸࡚ࡶ᭱ ࡶຠ⋡ࡢࡼ࠸ࣥࣔࢽࡢᏛྜᡂ᪉ἲ࡛࠶ࡾࠊᖺ㛫 1 ൨ 8,700 ࢺࣥࡢࣥࣔࢽ ែ❅⣲ࡀᮏἲ࡛⏕⏘ࡉࢀ࡚࠸ࡿ(Galloway et al., 2008)ࠋ⏕⏘ຠ⋡ࡢⰋ࠸ᑠ㯏ࡢ ᱂ᇵࡣ❅⣲ศࢆྵࡴ㔞ࡢ⫧ᩱࡢ౪⤥ࡀྍḞ࡛࠶ࡿࡀࠊᙜࡢせ࡞❅⣲ ⫧ᩱࡣ༡⡿➼࡛᥇᥀ࡉࢀࡿ◪▼㢗ࡗ࡚࠾ࡾࠊᑠ㯏ࡢ㔞⏕⏘ࡣ㞴ࡋࡗࡓࠋࣁ ࣮ࣂ࣮࣭࣎ࢵࢩࣗἲࡼࡾྜᡂࡉࢀࡓࣥࣔࢽࢆ⫧ᩱࡋ࡚⏝࠸ࡿࡇ࡛ࠊࡑ ࡢࡲࡲ࡛ࡣ⪔స㐺ࡉ࡞࠸⑭ࡏࡓᅵᆅ࠾࠸࡚ࡶᑠ㯏➼ࡢ✐≀ࡢ⏕⏘ࡀྍ⬟ ࡞ࡾࠊୡ⏺ࡢᛴ⃭࡞ேཱྀቑຍ♫ࡢⓎᒎ㈉⊩ࡋࡓࠋࡇࡢຌ⦼ࡼࡾࠊHaber ࡣ✵ẼỈࡽࣃࣥࢆసࡗࡓ⏨ࡋ࡚ࡶ▱ࡽࢀ࡚࠾ࡾࠊ1918 ᖺࣀ࣮࣋ࣝᏛ ㈹ࢆཷ㈹ࡋ࡚࠸ࡿࠋ1931 ᖺࡣ Bosch ࡶࠕ㧗ᅽᏛᛂࡢ◊✲ࠖࡼࡾࣀ࣮࣋ ࣝᏛ㈹ࢆཷ㈹ࡋ࡚࠸ࡿࠋᮏἲࡣ㧗ᅽࢆ⏝ࡋࡓᏛࣉࣟࢭࢫࡋ࡚Ṕྐⓗ ᭱ࡶ᭷ྡ࡛࠶ࡾࠊࡘே㢮ࡶࡓࡽࡋࡓ㈉⊩ࡶࡁ࠸ࡶࡢ࡛࠶ࡿ(㔜ᯇ, 2013)ࠋ 19 ୡ⣖ᚋ༙࡞ࡿᚤ⏕≀ࡢ㧗ᅽຠᯝࡘ࠸࡚◊✲ࡀ㐍ࢇࡔࠋRegnard ࡣ ⣙ 300 MPa ࡢ㧗ᅽࢆⓎ⏕ࡉࡏࡿᅽຊ⨨ࡢ㛤Ⓨᡂຌࡋࡓࠋ1884 ᖺࠊᙼࡣࡇࡢ 㧗ᅽ⨨ࢆ⏝࠸࡚ 6,000 m ࡢ῝ᾏྠ⛬ᗘࡢᅽຊ᮲௳(⣙ 60 MPa)࠾࠸࡚ୡ⏺ ࡛ึࡵ࡚ࣅ࣮ࣝ㓝ẕࡢᇵ㣴ࢆヨࡳࡓ(Regnard, 1884)ࠋࡑࡢ⤖ᯝࠊ㧗ᅽ᮲௳ୗ࠾ ࠸࡚ࡶ㓝ẕࡢ࢚ࢱࣀ࣮ࣝⓎ㓝ࡀᘬࡁ㉳ࡇࡉࢀࡿࡇࢆⓎぢࡋࡓࠋᙼࡢ◊✲ࡣ Buchner ࡼࡿ↓⣽⬊࡛ࡢⓎ㓝ࡢⓎぢ㛫᥋ⓗࡘ࡞ࡀࡿࠋBuchner ࡣࠊ1897 ᖺ 6 40-50 MPa ࡢᅽຊ᮲௳࡛㓝ẕࢆᅽᦢࡋ࡚ᚓࡓ㓝ẕᢳฟᾮࡀⅣỈ≀ࢆⓎ㓝ࡍ ࡿࡇࢆሗ࿌ࡋࡓࠋᙼࡣࠊⓎ㓝ᕤ⛬࠾࠸࡚⏕Ꮡࡋ࡚࠸ࡿᚤ⏕≀ࡀᚲࡎࡋࡶᚲせ ࡛ࡣ࡞ࡃࠊ㓝ẕࡢ⏕⏘ࡍࡿࢳ࣐࣮ࢮ(㓝⣲)ࡀⓎ㓝㔜せ࡛࠶ࡿࡇࢆⓎぢࡋࡓࠋ 1907 ᖺࠕ↓⣽⬊࡛ࡢⓎ㓝ࡢⓎぢ⏕Ꮫࡼࡿ◊✲ࠖࡢᡂᯝࡼࡾࠊࣀ࣮࣋ ࣝᏛ㈹ࢆཷ㈹ࡋࡓࠋᙼࡣ㧗ᅽ᮲௳ୗ࠾ࡅࡿ㓝⣲Ꮫࡢඛ㥑⪅ࡋ࡚▱ࡽࢀ࡚ ࠸ࡿ(Jaenicke, 2007)ࠋ Roger ࡣࠊ1892 ᖺ㧗ᅽ᮲௳࠾ࡅࡿ⣽⳦ࡢάᛶࡘ࠸࡚ࡢ 2 ಶࡢ㔜せ ࡞ሗ࿌ࢆࡋࡓ(Roger, 1892; Roger, 1895)ࠋ1 ಶ┠ࡣࠊ㧗ᅽ᮲௳ୗࡢάᛶᣲືࡀ ᚤ⏕≀ࡢᒓ✀ࡼࡗ࡚␗࡞ࡿ࠸࠺ሗ࿌࡛࠶ࡿࠋ࠼ࡤࠊStaphylococcus aureus ࡣ 300 MPa ⛬ᗘ࡛ࡣࡁ࡞ᙳ㡪ࢆཷࡅ࡞࠸ࡀࠊStreptococcus ᒓࡢ⣽⳦ࡣྠᅽຊ ᮲௳࡛ 30%⛬ᗘࡢ⏕⳦ᩘࡢపୗࡀㄆࡵࡽࢀࡿࠋ2 ಶ┠ࡣࠊᰤ㣴⣽⬊ⱆ⬊ࡢᅽຊ ⪏ᛶࡢ㐪࠸࡛࠶ࡿࠋBacillus anthracis ࡢⱆ⬊ࡣᰤ㣴⣽⬊ࡢሙྜࡼࡾࡶᅽຊ⪏ᛶࡀ 㧗࠸ࡇࢆሗ࿌ࡋࡓࠋࡇࢀࡽࡢሗ࿌ࡣࠊ㧗ᅽࡼࡗ࡚ᚤ⏕≀ࡀάᛶࡍࡿࡇ ࢆึࡵ࡚♧ࡋࡓሗ࿌࡛࠶ࡾࠊᙼࡢⓎぢ௨᮶ࠊᵝࠎ࡞ᚤ⏕≀✀ࡢᅽຊ⪏ᛶࡀ◊✲ࡉ ࢀࠊ㔜せ࡞▱ぢࡀ✚ࡉࢀ࡚࠸ࡿࠋ 7 ➨ 4 㡯 㧗ᅽ◊✲ࡢṔྐ㸫㣗ရࡢ㧗ᅽຍᕤࡢᛂ⏝㸫 ୡ⏺࡛ึࡵ࡚㣗ရࡢ㧗ᅽฎ⌮ࢆሗ࿌ࡋࡓࡢࡣ Hite (1899 ᖺ)࡛࠶ࡿࠋᙼࡣࠊ ࣑ࣝࢡࡢ㛗ᮇಖᏑࡢࡓࡵ⇕ẅ⳦௨እࡢࣉ࣮ࣟࢳࢆ⪃ࡋࡓ᭱ึࡢே≀࡛࠶ ࡿࠋ463 MPaࠊ1 h ࡢ᮲௳࡛㧗ᅽฎ⌮ࡉࢀࡓ࣑ࣝࢡࡣࠊᑡ࡞ࡃࡶ 24 h ࡣ㓟ᛶ ࡀᢚไࡉࢀ࡚⏑ࡀಖᣢࡉࢀࡿࡇࢆⓎぢࡋࡓࠋࡲࡓࠊᙼࡣ㧗ᅽฎ⌮୰ࡢ ᗘ (50~80°C)ࡢᙳ㡪ࡶホ౯ࡋ࡚࠸ࡿ(Hite et al., 1914)ࠋ Bridgman ࡣࠊࡇࢀࡲ࡛ࡼࡾࡶ 10 ಸ௨ୖ㧗࠸㧗ᅽࢆⓎ⏕ࡉࡏࡿ㧗ᅽ⨨ࢆ㛤Ⓨ ࡋࡓࠋࡑࢀࡼࡾ 1912 ᖺ㧗ᅽୗ࠾ࡅࡿỈࡢ≧ែኚࡢ┦㛵ᅗࢆሗ࿌ࡋࡓ (Bridgman, 1912)ࠋ1914 ᖺࡣࠊ㭜༸ 500~700 MPaࠊ30~60 min ࡢ᮲௳࡛㧗ᅽฎ ⌮ࡍࡿࡇࡼࡾࠊ༸Ẇࢆࡿࡇ࡞ࡃࠊ༸㯤࣭༸ⓑࡀจᅛࡍࡿࡇࢆⓎぢࡋࡓ (Bridgman, 1914)ࠋᙼࡢሗ࿌ࡣ㧗 ࡛ほᐹࡉࢀࡿࢱࣥࣃࢡ㉁ࡢኚᛶࡀ㠀⇕ⓗ᮲௳ ࡢ㧗ᅽ࡛ࡶྠᵝᘬࡁ㉳ࡇࡉࢀࡿࡇࢆึࡵ࡚᫂ࡽࡋࡓ⏬ᮇⓗ࡞Ⓨぢ࡛࠶ ࡾࠊࡑࡢᚋࡢࢱࣥࣃࢡ㉁ࡢ㧗ᅽኚᛶ࣓࢝ࢽࢬ࣒㛵ࡍࡿ◊✲ࡢඛ㥑ࡅ࡞ࡗࡓࠋ Bridgman ࡣࠊ ࠕ㉸㧗ᅽ⨨ࡢ㛤Ⓨࡑࢀࡼࡿ㧗ᅽ≀⌮Ꮫ㛵ࡍࡿⓎぢࠖࡼࡾ 1946 ᖺࣀ࣮࣋ࣝ≀⌮Ꮫ㈹ࢆཷ㈹ࡋࠊ㧗ᅽ≀⌮Ꮫࡢ∗ࡤࢀ࡚࠸ࡿࠋ ࡋࡋࠊHite ࡸ Bridgman ࡢ㧗ᅽฎ⌮ࡼࡗ࡚㣗ရࡢ㢼ࡸᰤ㣴౯ࢆ⇕ኚᛶ࡛ ᦆ࡞ࢃࡎ㣗ရࡢಖᏑᛶࢆྥୖࡉࡏࡿ⏬ᮇⓗ࡞Ⓨぢࡣࠊᐙᗞ⏝෭ⶶᗜࡢᬑཬࡸ ịࡢᕤᴗⓗ⏕⏘➼ࡢ᪂つ෭ⶶᢏ⾡ࡢ㛤Ⓨࡼࡾ㣗ရ⏘ᴗ⏺㢳ࡳࡽࢀࡿࡇࡣ 8 ࡞ࡗࡓ(㕥ᮌ, 2013)ࠋ 㣗ရࡢ㧗ᅽ⏝ࡢ㌿ᶵ࡞ࡗࡓࡇࡀ 1968 ᖺ㉳ࡇࡗࡓ◊✲⏝₯ỈⰄ Alvin ྕࡢỿἐ௳࡛࠶ࡿ(Pope, 1973)ࠋAlvin ྕࡣ Woods Hole Oceanographic Institution (WHOI)ᡤᒓࡍࡿ₯ỈⰄ࡛࠶ࡿࠋ1968 ᖺࡢࠊAlvin ྕࡣᅇ⯟㏵୰ ᨾ㐼㐝ࡋࡓࠋᖾ࠸ࡶဨࡣဨ⬺ฟ࡛ࡁࡓࡀ⯪యࡣ 1,543 m ࡢ῝ᾏỿࢇ ࡔࠋAlvin ྕࡀ῝ᾏࡽᘬࡁᥭࡆࡽࢀࡓࡢࡣࠊࡑࡢᨾࡽ 10 ᭶ᚋ࡛࠶ࡿࠋࡑ ࡢᚋࡢ⯪యࡢㄪᰝ࠾࠸࡚ࠊ㦫ࡃࡁࡇࡀ᫂ࡽ࡞ࡗࡓࠋ⯪యඹ῝ᾏ ỿࢇ࡛࠸ࡓࣜࣥࢦࡸࢧࣥࢻ࢘ࢵࢳ➼ࡢእぢࠊࠊ㤶ࡾࡀỿἐࡍࡿ๓ࢇ ኚࡋ࡚࠸࡞ࡗࡓࡢ࡛࠶ࡿࠋࡑࢀࡔࡅ࡛ࡣ࡞ࡃࠊᚤ⏕≀Ꮫⓗ࣭⏕Ꮫⓗࡶ㣗 ရࡢရ㉁ࡀⰋࡃಖᣢࡉࢀ࡚࠸ࡓࡇࡀ᫂ࡽ࡞ࡗࡓࠋ୍⯡ⓗࡣࠊ4°C ⛬ᗘࡢ ෭ⶶ᮲௳࠾࠸࡚ࡣࠊࢹࣥࣉࣥࡸࢱࣥࣃࢡ㉁➼ࡣᩘ㐌㛫࡛ຎࡋ࡚⭉ᩋࡍࡿࠋ Alvin ྕࡢ⯪య࡛ಖᏑࡉࢀ࡚࠸ࡓ㣗ရࡢရ㉁ࡣࠊ῝ᾏ≉᭷ࡢప ࠊ㈋ᰤ㣴᮲௳ࡢ ᆅ⾲ࡢẼᅽ(⣙ 0.1 MPa)ẚ㍑ࡋ࡚ 100 ಸ௨ୖ㧗࠸ 15 MPa ⛬ᗘࡢᅽຊ ࡼࡗ࡚⥔ᣢࡉࢀ࡚࠸ࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋࡇࡢ▱ぢࡣࠊ㧗ᅽ᮲௳ࡀ⭉ᩋࢆᘬࡁ ㉳ࡇࡍᚤ⏕≀ࡢ௦ㅰάᛶࢆᢚไࡍࡿࡶ㛵ࢃࡽࡎࠊ㣗ရࡢရ㉁ࢆຎࡉࡏ࡞࠸ ࡇࢆ♧ࡋ࡚࠾ࡾࠊ㣗ရࡢప ẅ⳦ࡢ㧗ᅽࡀ⏝࡛ࡁࡿࡇࢆ♧၀ࡋ࡚࠸ࡿࠋ Alvin ྕࡢỿἐᨾ௨᮶ࠊ㧗ᅽᢏ⾡ࡢ㣗ရຍᕤࡢᛂ⏝ࡢྍ⬟ᛶࡀ◊✲ࡉࢀࠊ 1987 ᖺᯘ ຊࡼࡾ㣗ရຍᕤࡢ㧗ᅽ⏝ࡀᥦၐࡉࢀࡓ(Hayashi et al., 1987)ࠋ 9 㧗ᅽࡣࠊ⏕యศᏊࡢ㠀ඹ᭷⤖ྜࡢࡳస⏝ࡍࡿࡓࡵࠊձ㣗ရ⣲ᮦࡀᣢࡘ᪂㩭࡞ 㢼ࠊⰍࠊࢃ࠸ࢆಖᣢ࡛ࡁࡿࠊղຍ⇕ࡼࡾ◚ቯࡉࢀࡿࣅࢱ࣑ࣥ C ➼ࡢ᭷⏝ ᡂศࡢຎࡀᑡ࡞࠸ࠊճຍ⇕ࡼࡿࢡ࣑ࣜࣝࢻ➼ࡢ␗ᖖ≀㉁ࡸ␗⮯ࡀⓎ⏕ ࡋ࡞࠸ࠊմຍ⇕ຍᕤࡣ␗࡞ࡿ⊂≉࡞≀ᛶࡀ⏕ࡌࡿࠊյຍ⇕ฎ⌮ẚ࡚┬࢚ࢿ ࣝࢠ࣮࡛࠶ࡿ➼ࡢⅬࡀ࠶ࡿ(ᯘ, 1991)ࠋࠕ㣗ရຍᕤࡢ㧗ᅽ⏝ࠖࡢᥦၐ௨㝆ࠊ ከࡃࡢ◊✲⪅ࡼࡗ࡚㧗ᅽᢏ⾡ࢆᛂ⏝ࡋࡓప ẅ⳦ᢏ⾡ࠊ㣗ရຍᕤ࣭〇㐀ᢏ⾡ࠊ 㓝⣲άᛶࡸࡑࢀࡼࡿ᭷⏝ᡂศࡢቑᙉ➼ࡢ◊✲ࡀ᥎㐍ࡉࢀࡓࠋ ᪥ᮏᅜෆ࡛ࡣ㎰ᯘỈ⏘┬ࡼࡾࠕ㣗ရ⏘ᴗ㉸㧗ᅽ⏝ᢏ⾡◊✲⤌ྜࠖࡀ⤌⧊ࡉ ࢀࠊ ࠕ㣗ရ⏘ᴗࡢᮍ᮶ࢆᣅࡃ㸫㧗ᅽᢏ⾡㧗ᐦᗘᇵ㣴ࠖ࠸࠺ㄢ㢟࡛ࣉࣟࢪ࢙ࢡ ࢺ(ᮌᮧ, 1993)ࡀጞࡲࡾࠊ⏘Ꮫᐁࡀ୍య࡞ࡗࡓ㧗ᅽᢏ⾡ᇶ࡙ࡃ᪂ࡓ࡞㣗ရຍ ᕤᢏ⾡ࡢ◊✲࣭㛤Ⓨࢆ㐍ࡵࡓ(Kasuga, 1998)ࠋ᪥ᮏᅜෆ࡛᭱ࡶ㧗ᅽ◊✲ࡀάⓎ ⾜ࢃࢀࡓ㒔㐨ᗓ┴ࡢ 1 ࡘࡣ᪂₲┴࡛࠶ࡿࠋ1989 ᖺ㧗ᅽ㣗ရຍᕤࡢᐇ⏝ࢆ┠ ᣦࡋࡓࠕ㉸㧗ᅽᢏ⾡ࡢ㣗ရ➼ࡢᛂ⏝㛵ࡍࡿ◊✲ (᪂₲┴㧗ᅽᛂ⏝㣗ရ◊✲ )ࠖࡀⓎ㊊ࡋࠊࡑࡢ◊✲ᡂᯝࡣࠕ㧗ᅽ⏝㛵ࡍࡿ◊✲ᡂᯝሗ࿌᭩ࠖࡋ࡚Ⓨ หࡉࢀࡓ(1991)ࠋ㎰ᯘỈ⏘┬ࡢ㧗ᅽ㛵㐃ࣉࣟࢪ࢙ࢡࢺࡢ⤊ᚋࡶࠊ᪂₲┴࡛ࡣ 2003 ᖺ⤒῭⏘ᴗ┬ࡢබເᆺጤク◊✲ᴗࠕᆅᇦ᪂⏕ࢥࣥࢯ࣮ࢩ࣒㸫㧗ᅽฎ ⌮ࢆ⏝ࡋࡓ᪂つᶵ⬟ᛶ㣗ᮦࡢ㛤Ⓨ⏘ᴗࠖࡀ᥇ᢥࡉࢀࠊ2007 ᖺࡣ㧗ᅽᢏ ⾡㛵ࡍࡿᡓ␎ⓗ࡞ᶆ‽ࢆ᥎㐍ࡍࡿࡓࡵࡢࠕ㧗ᅽᇶ┙ᢏ⾡ᶆ‽ᶵᵓࠖࢆ❧ࡕ 10 ୖࡆ࡚࠸ࡿࠋ2008 ᖺࡣࠊ᪥ᮏ⛉Ꮫᢏ⾡⯆ᶵᵓ (JST) 2007 ᖺᗘࠕ᪂₲┴ᆅᇦ ⤖㞟ᆺ◊✲ࣉࣟࢢ࣒ࣛ㸫㣗ࡢ㧗ຍ౯್㈨ࡍࡿᇶ┙ᢏ⾡ࡢ㛤Ⓨࠖࢆ㛤ጞࡋࠊ ࡑࡢᡂᯝࡣࡑࡢᡂᯝࢆࠕ㐍ࡍࡿ㧗ᅽ㣗ရຍᕤᢏ⾡ࠖ㢟ࡋ࡚Ⓨหࡋࡓࠋ᪂₲┴ ࠾ࡅࡿ㧗ᅽຍᕤ㣗ရ㛤ⓎࡢṔྐࡣࠊ㕥ᮌ(2011)ࡼࡗ࡚ࡲࡵࡽࢀ࡚࠸ࡿࠋ 㧗ᅽ◊✲ࡢᡂᯝࢆⓎ⾲ࡋ࡚㆟ㄽࡋྜ࠺ሙࡋ࡚ࠊ⏕≀㛵㐃㧗ᅽ◊✲ࡀ 1988 ᖺタ❧ࡉࢀࡓࠋྠᖺ➨ 1 ᅇࢩ࣏ࣥࢪ࣒࢘ࡀி㒔࡛ࠊ2013 ᖺࡣ➨ 18 ᅇࢩࣥ ࣏ࢪ࣒࢘ࢆᒱ㜧Ꮫ࡛㛤ദࡍࡿࡇࡀ࡛ࡁࡓࠋ2015 ᖺࡣᗈᓥ࡛➨ 20 ᅇࢩ࣏ࣥ ࢪ࣒࢘ࡀ㛤ദࡉࢀࡿணᐃ࡛࠶ࡿࠋᅜእ┠ࢆྥࡅࡿ International Conference on High Pressure Bioscience and Biotechnology (HPBB)ࡀ 2000 ᖺタ❧ࡉࢀࠊ௨㝆 2 ᖺẖୡ⏺ྛᆅ࡛㛤ദࡉࢀ࡚࠸ࡿ(Nomura and Iwahashi, 2014)ࠋ2014 ᖺ➨ 8 ᅇ ࡀࣇࣛࣥࢫ࡛㛤ദࡉࢀࡓࠋ2016 ᖺࡣ➨ 9 ᅇࡀ࢝ࢼࢲ࡛㛤ദࡉࢀࡿண ᐃ࡛࠶ࡿࠋ 11 ➨ 2 ⠇ 㓝ẕ 㓝ẕ(Yeast)ࡣࠊ┤ᚄ 5~10 ȣm ⛬ᗘࡢ༸ᆺ࡛ࠊฟⱆࡸศࡼࡗ࡚ቑṪࡍࡿ┿ ᰾༢⣽⬊⏕≀࡛࠶ࡿࠋ㓝ẕࡣࣄࢺྠࡌࡼ࠺ᑑࡶᏑᅾࡍࡿࠋ㟁Ꮚ㢧ᚤ㙾࡛ ฟⱆ㓝ẕࢆほᐹࡍࡿࠊ⣽⬊ࡢ⾲㠃ፉ㓝ẕࡀฟⱆࡍࡿ⏕ࡌࡓฟⱆࠊ⮬㌟ ࡀฟⱆࡋࡓ⏕ࡌࡓฟ⏕ࡤࢀࡿ」ᩘࡢพฝࡀ☜ㄆ࡛ࡁࡿ(㔝ᮧ, ᒾᶫ, 2013)ࠋࡇࡢฟⱆࡀ⣽⬊ࡢ⾲㠃ṧࡿࡓࡵࠊࡑࡢᩘࡀฟⱆᅇᩘࠊ༶ࡕ㓝ẕࡢᑑ ┦ᙜࡍࡿࠋ⣙ 20 ᅇฟⱆࡍࡿࠊࡑࢀ௨ୖࡣฟⱆࡍࡿࡇࡀ࡛ࡁ࡞ࡃ࡞ࡿࡇ ࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Egilmez and Jazwinski, 1989)ࠋฟⱆᅇᩘࡣ⏕⌮ⓗ࡞せᅉ࡛ᙳ 㡪ࢆཷࡅࡿࡇࡶ࠶ࡾࠊ⎔ቃࢫࢺࣞࢫ➼࡛ᐜ᫆ኚࡍࡿࠋࡲࡓࠊ㓝ẕࡣ〇ࣃࣥ ࡸ㔊㐀➼ࡢ㣗ရ⏘ᴗศ㔝ᗈࡃ⏝࠸ࡽࢀ࡚࠸ࡿࡔࡅ࡛࡞ࡃࠊࣂ࢜ࢸࢡࣀࣟࢪ ࣮ศ㔝࠾࠸࡚ࡶࣂ࢚࢜ࢱࣀ࣮ࣝࡢ⏕⏘➼㔜せ࡞ᙺࢆᯝࡓࡋ࡚࠸ࡿ (Bravim, 2012)ࠋ 12 ➨ 1 㡯 ࣔࢹࣝ⏕≀ࡋ࡚ࡢ㓝ẕ ᭱ࡶ୍⯡ⓗ࡞㓝ẕ࡛࠶ࡿ Saccharomyces cerevisiae ࡣࠊࣄࢺྠࡌ┿᰾⏕≀࡛ ࠶ࡿୖࢤࣀ࣒ᵓ㐀ࡀẚ㍑ⓗ༢⣧࡛࠶ࡿࡢ࡛ࠊ⭠⳦➼ࡢཎ᰾⏕≀࡛ᚓࡓ▱ぢ ẚ㍑ࡋ࡚ࠊ㓝ẕࡢศᏊ⏕≀Ꮫⓗ▱ぢࢆࣄࢺᛂ⏝ࡋ᫆࠸࠸࠺Ⅼࡀ࠶ࡿࠋࡲ ࡓࠊୡ௦㛫ࡀ▷ࡃࠊᏳ౯࡞ᇵᆅ࡛ቑṪ࡛ࡁࠊẘᛶࡶ↓ࡃࠊ⏕⌮≧ែࡢ㧗࠸⌧ ᛶࡀᮇᚅ࡛ࡁࡿࡓࡵᏛ➼ࡢᐇ㦂ᐊࡢタഛ࡛ẚ㍑ⓗ⡆༢◊✲࡛ࡁࡿࠋࡇࢀ ࡽࡢⅬࡽ㓝ẕࡣศᏊ⏕≀Ꮫࢆጞࡵᵝࠎ࡞ศ㔝ࡢࣂ࢜ࢵࢭࡢࣔࢹࣝ⏕ ≀ࡋ࡚⏝ࡉࢀ࡚࠸ࡿ(Haney et al., 2001; Matsuoka et al., 2005; Iwahashi et al., 2007; Yasokawa and Iwahahsi, 2010)ࠋ 1996 ᖺࡣࠊ┿᰾⏕≀ࡋ࡚ึࡵ࡚ S. cerevisiae ࡢࢤࣀ࣒ࡢሷᇶ㓄ิሗࡀ ゎㄞࡉࢀࡓ(Goffeau et al.,1996)ࠋ㓝ẕࡢ⣙ 6000 ✀㢮ࡢ㑇ఏᏊࢥ࣮ࢻࡉࢀ࡚࠸ ࡿࢱࣥࣃࢡ㉁ࡢ࠺ࡕࠊ࠾ࡼࡑ 80%ࡀ⏕Ꮡᚲ㡲࡛࡞࠸ࡇࡶ᫂ࡽ࡞ࡗ࡚࠸ ࡿࠋࡲࡓࠊ㓝ẕࢤࣀ࣒ࡼࡗ࡚ࢥ࣮ࢻࡉࢀ࡚࠸ࡿࢱࣥࣃࢡ㉁ࡢᑡ࡞ࡃࡶ 31% ࡣࣄࢺ࣍ࣔࣟࢢࢆᣢࡗ࡚࠾ࡾࠊ㏫㑇ఏ㛵㐃ࡍࡿࣄࢺࢤࣀ࣒ࡢ⣙ 50%ࡢ 㑇ఏᏊࢆࠊ㓝ẕࡣ࣍ࣔࣟࢢࡋ࡚ᣢࡗ࡚࠸ࡿࡇࡶሗ࿌ࡉࢀ࡚࠸ࡿ(Hartwell, 2004)ࠋࡍ࡞ࢃࡕࠊ㓝ẕࣄࢺࡢࢱࣥࣃࢡ㉁ࡢከࡃࡣྠᵝᶵ⬟ࡋ࡚࠾ࡾࠊࢫࢺ ࣞࢫᛂ⟅࣓࢝ࢽࢬ࣒ゎ᫂ࡢࡓࡵࡢࣔࢹࣝ⏕≀ࡋ࡚ࡶ◊✲⏝ࡉࢀ࡚࠸ࡿ (Haney et al., 2001; Hohmann, 2002, Iwahashi et al., 2003; Iwahashi et al., 2005)ࠋࡲ 13 ࡓࠊ㓝ẕࡢࢤࣀ࣒ゎᯒᚋࠊ㑇ఏᏊࢆᦚ㍕ࡋࡓ DNA ࢳࢵࣉࡀ᪩ᛴ౪⤥ࡉࢀ ࡓ(ᒾᶫ, 2002)ࠋࡑࢀᨾࢤࣀ࣑ࢡࢫゎᯒ⤖ᯝࡢ✚ࡀ㇏ᐩ࠶ࡾࠊゎᯒࢆᐜ᫆ ࡋ࡚࠸ࡿࠋ㓝ẕࢤࣀ࣑ࢡࢫࡢ୍␒ࡁ࡞Ⅼࡣࠊྛ㑇ఏᏊࡢᶵ⬟ሗࡢ㇏ᐩࡉ ࡛࠶ࡾࠊࡉࡽゎᯒࢆᐜ᫆ࡋ࡚࠸ࡿࠋ 㓝ẕࣄࢺࡢ྾ᶵ⬟㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡣẚ㍑ⓗࡼ ࡃಖᏑࡉࢀ࡚࠾ࡾࠊ࢚ࢿࣝࢠ࣮⏕⏘ࡢࡓࡵࡢ㓟ⓗࣜࣥ㓟ࡶྠᵝࡢᶵ⬟ࢆ᭷ ࡍࡿࠋຍ࠼࡚ࠊ㓝ẕࡣ࢚ࢱࣀ࣮ࣝⓎ㓝ࡼࡾቑṪࡍࡿࡇࡀ࡛ࡁࡿࡢ࡛ࠊ࣑ࢺࢥ ࣥࢻࣜࡀኚ␗ࡋ࡚྾ᶵ⬟ࡀḞኻࡋ࡚ࡶ⏕Ꮡࡍࡿࡇࡀྍ⬟࡛࠶ࡿࠋࡑࡢࡓ ࡵ࣑ࢺࢥࣥࢻࣜࡀ㛵㐃ࡍࡿ㑇ఏࡸ⒴ࢆጞࡵࡋࡓከࡃࡢẼࡸ྾㜼ᐖ ➼ᑐࡍࡿᇶ♏◊✲ࡢࣔࢹࣝ⏕≀ࡶ࡞ࡗ࡚࠸ࡿ (⚟ཎ, 1986)ࠋ ㏆ᖺࠊ㓝ẕࡀ⎔ቃ୰ᬑ㐢ⓗᏑᅾࡍࡿ⏕≀ࡢ 1 ࡘ࡛࠶ࡿࡇࡀ᫂ࡽࡉ ࢀࠊ㓝ẕࡢࢺࢥࢩࢥࢤࣀ࣑ࢡࢫࡀ⏕ែẘᛶホ౯ᛂ⏝࡛ࡁࡿࡇࡀሗ࿌ࡉࢀࡓ (Liti et al., 2009)ࠋࡇࢀࡼࡾࠊ⎔ቃ୰ࡢṧ␃㎰⸆ࡸ㔠ᒓࢼࣀ⢏Ꮚ➼ࡢ⏕యࡢẘ ᛶホ౯㓝ẕࢆ⏝ࡍࡿࡇࡀྍ⬟࡞ࡗࡓࠋ 14 ➨ 2 㡯 Ⓨ㓝㣗ရࡋ࡚ࡢ㓝ẕ ᡃࠎே㢮ࡣࠊᚤ⏕≀Ⓨ㓝࠸࠺ᴫᛕࢆ⌮ゎࡋ࡚࠸࡞࠸ྂ௦ࡽఏ⤫ⓗⓎ 㓝㣗ရࢆ⏕⏘ࡋ࡚ࡁࡓࠋࢪ࡛ࡣࠊᑠ㯏(Okada et al., 1992)ࠊ࠺ࡶࢁࡇࡋ (Adegoke and Babalola, 1988)ࠊ࢟ࣕࢵࢧࣂ(Oyewole and Odunfa, 1988)ࠊࢯ࣒ࣝ࢞ (Mohammed et al., 1991)➼ࡢᑠ㯏⢊Ⓨ㓝㣗ရࡀᵝࠎ࡞ᚤ⏕≀ࡼࡗ࡚⏕⏘ࡉࢀ࡚ ࠸ࡿࠋ࣮ࣚࢢࣝࢺࡸࢳ࣮ࢬ(Mitsuoka et al., 2002)ࠊ㤿ங㓇(Ishii, 2002)➼ࡢⓎ㓝ங 〇ရࡣங㓟⳦ࡼࡗ࡚⏕⏘ࡉࢀࡿࠋᮏ◊✲࡛⏝࠸ࡿ S. cerevisiae ࡣࠊࣝࢥ ࣮ࣝⓎ㓝ࡼࡾ࣡ࣥࠊࣅ࣮ࣝࠊΎ㓇➼ࡢ㓇㢮ࠊჯࠊ㓺Ἔ➼ࡢㄪᩱࠊࣃࣥࠊ ₕ≀➼ࡢᵝࠎ࡞Ⓨ㓝㣗ရࢆ⏕⏘ࡍࡿ᭷⏝ᚤ⏕≀ࡋ࡚ᗈࡃ▱ࡽࢀ࡚࠸ࡿ (Nomura and Iwahashi, 2014)ࠋࡇࢀࡽࡢఏ⤫ⓗ࡞Ⓨ㓝㣗ရࡣ㣗ࡋ࡚ࡔࡅ࡛ࡣ ࡞ࡃࠊᢠ㓟άᛶࡸᢠࣞࣝࢠ࣮ࠊච㈿ά➼ࡢ᭷┈࡞ຠᯝࡀᮇᚅ࡛ࡁࡿᗣ㣗 ရࡸࢥ࣑ࣗࢽࢣ࣮ࢩࣙࣥࢆࡍࡿႴዲရࡋ࡚ࡶ㣗ࡉࢀ࡚࠸ࡿ(Yang et al., 2010) ࠋ㓝ẕࡣⓎ㓝㣗ရࡢ⏕⏘ᙺ❧ࡘ୍᪉ࠊࣇ࣮ࣝࢶࢪ࣮ࣗࢫࡢࡼ࠺࡞㣗ရࡢ ⭉ᩋࡸ࣒࢟ࢳ➼ࡢ㐣Ⓨ㓝ࢆᘬࡁ㉳ࡇࡋࠊ㣗ရࡢရ㉁ࢆຎࡉࡏ࡚㔜࡞⤒῭ⓗ ᦆ ኻ ࢆ ᘬ ࡁ ㉳ ࡇ ࡍ ࡇ ࡶ ▱ ࡽ ࢀ ࡚ ࠸ ࡿ (Basak et al., 2002; Lee et al., 2003; Patrignani et al., 2009)ࠋ 15 ➨ 3 ⠇ 㧗ᅽ㣗ရຍᕤᢏ⾡ 㧗ᅽ㣗ရຍᕤᢏ⾡ࡣࠊᩘⓒ MPa ࡢ㧗ᅽ᮲௳ࡼࡾ㣗ရࡢẅ⳦ࡸᶵ⬟ᛶᡂศ ࡢᐩࠊ㓝⣲άᛶࡢྥୖࠊຍ౯್ࡢ➼ࢆ┠ⓗ㠀⇕ⓗ࠶ࡿ࠸ࡣຍ⇕⤌ࡳ ྜࢃࡏࡿࡇࡼࡾࠊ㣗ရࢆຍᕤࡍࡿᢏ⾡ࡢ⥲⛠࡛࠶ࡿࠋ1987 ᖺி㒔Ꮫᯘ ຊ ຓᩍᤵ(ᙜ)ࡼࡾ㧗ᅽᢏ⾡ࢆ㣗ရຍᕤᛂ⏝ࡍࡿࡇࢆᥦၐࡉࢀࠊᮏᢏ ⾡ࡢ◊✲ࡀጞࡲࡗࡓࠋ ➨ 1 㡯 㧗ᅽຍᕤ㣗ရࡢ㛤Ⓨၥ㢟Ⅼ 㧗ᅽᢏ⾡ࡣࣅࢱ࣑ࣥ C ➼ࡢ㣗ရ୰ࡢ᭷⏝ᡂศࡸ᪂㩭࡞㢼➼ࢆ⇕ኚᛶ࡛◚ ቯࡍࡿࡇ࡞ࡃࠊ㣗ရࡢຍᕤᛂ⏝࡛ࡁࡿ࠸࠺Ⅼࡀ࠶ࡿࠋࡑࡢࡓࡵ㧗ᅽ㣗 ရຍᕤࡣࠊ㠀⇕ⓗ࠶ࡿ࠸ࡣᚑ᮶ࡢຍ⇕ຍᕤࡼࡾࡶప࠸ ᗘ࡛㣗ရࢆຍᕤࡋࠊᚤ ⏕≀ࢆάᛶࡍࡿࡇࡀྍ⬟࡛࠶ࡿࠋ㧗ᅽ㣗ရຍᕤࡣࠊࡇࢀࡲ࡛ຍ⇕ࡢ௦᭰ 㧗ᅽຍᕤࢆ⏝ࡋࡓ㧗ᅽຍᕤ⏕ࢪ࣒ࣕ (ᇼỤࡽ, 1991)ࡸ✐㢮ࡢ྾Ỉ㧗ᅽຍ ᕤࢆ⏝ࡋࡓ㞧✐ࡈࡣࢇࡸࣃࢵࢡࡈࡣࢇ(ᒣᓮ, ➲ᕝ, 2000)ࠊ㣗ရῧຍ≀ࡢ௦᭰ ࡋ࡚㧗ᅽẅ⳦ࡋࡓࣁ࣒ࡸࢯ࣮ࢭ࣮ࢪ(᳃, 㔜ஂ, 1990; ㎷⏣, 㕥ᮌ, 1990)ࠊ㧗 ᅽࡼࡿ∻⾃ࡢẆࡴࡁ(Murokoshi, 2004)➼ᛂ⏝ࡉࢀ࡚࠸ࡿࠋࡋࡋ࡞ࡀࡽࠊ㧗 ᅽᢏ⾡ࡼࡿᵝࠎ࡞㣗ရຍᕤࡸᚤ⏕≀ࡢάᛶࡣࠊ300 MPa ௨ୖࡢ㧗ᅽࡀ 16 ᚲせ࡛࠶ࡿࠋࡑࡢࡓࡵࡣ㧗ᅽ⪏࠼ࡿࡇࡀ࡛ࡁࡿ㧗ࢥࢫࢺ࡞⪏ᅽᐜჾࢆ᭷ ࡍࡿ㧗ᅽ⨨ࡀᚲ㡲࡛࠶ࡾࠊ㣗ရࡢ㧗ᅽຍᕤࡣ㧗ຍ౯್ࢆ᭷ࡍࡿ㣗ရ㝈 ࡽࢀ࡚࠾ࡾࠊ㧗ᅽᢏ⾡ࡢᬑཬࡀጉࡆࡽࢀ࡚࠸ࡿࡢࡀ⌧≧࡛࠶ࡿࠋ ➨ 2 㡯 Pressure Regulated Fermentation ㏆ᖺࠊ㣗ရࡢ㧗ᅽຍᕤࢆࡼࡾᬑཬࡉࡏࡿࡓࡵࠊࡼࡾప࠸ᅽຊࡼࡾ㣗ရࡢ Ⓨ㓝ࢆไᚚࡍࡿᢏ⾡(Pressure Regulated Fermentation; PReF)ࡢ㛤Ⓨࡀᥦࡉࢀ࡚ ࠸ࡿ(Nomura and Iwahashi, 2014)ࠋⓎ㓝㣗ရࢆ⏕⏘ࡍࡿ㓝ẕࡢⓎ㓝ࡀไᚚ࡛ࡁ࡞ ࠸ሙྜࠊㄪᾮࡢⓑ⃮ࡸᐜჾࡢ⭾ᙇ➼ࡢ㐣Ⓨ㓝ࢆᘬࡁ㉳ࡇࡋࠊ㔜࡞⤒῭ⓗᦆኻ ࢆ⏕ࡌࡿ༴㝤ᛶࡀ࠶ࡿࠋᚑ᮶ࡢⓎ㓝㣗ရࡢⓎ㓝ࢆไᚚࡍࡿ᪉ἲࡣࠊ୍⯡ⓗຍ⇕ ࡸ෭ⶶࠊሷࡸ㓟ࡢῧຍ࡛࠶ࡿࠋ≉ࠊங㓟⳦ࡢ⏘⏕ࡍࡿங㓟ࡣዃ㞧ᚤ⏕≀ࡢቑṪ ࢆᢚไࡍࡿࡓࡵࠊྂࡃࡽ㓝ẕඹⓎ㓝㣗ရࡢ⏕⏘⏝ࡉࢀ࡚ࡁࡓࠋPReF ᢏ⾡ࡣࠊ㧗ᅽᢏ⾡ࡢⅬࢆάࡋࠊⓎ㓝㣗ရ୰Ꮡᅾࡍࡿࣅࢱ࣑ࣥ C ➼ࡢ᭷⏝ ᡂศࢆ⥔ᣢࡋࡘࡘࠊᚤ⏕≀ࡢⓎ㓝ࢆไᚚࡍࡿࡇࢆ┠ᣦࡋ࡚࠸ࡿࠋࡲࡓࠊPReF ᢏ⾡ࢆᛂ⏝ࡍࡿࡇࡣࠊຍ⇕ࡼࡿ᭷⏝≀㉁ࡢຎࡔࡅ࡛ࡣ࡞ࡃࠊሷࡸ㓟ࡢ㐣 ῧຍࡼࡿᗣ⿕ᐖࡢⓎ⏕ࡶᢚไࡍࡿࡇࡀᮇᚅ࡛ࡁࡿࠋేࡏ࡚ࠊPReF ᢏ⾡࡛ ࡣࠊ㧗ᅽ⨨ࡢࢥࢫࢺࢆ๐ῶࡍࡿࡓࡵࠊ100~200 MPa ⛬ᗘࡢ✜ࡸ࡞୰㧗ᅽ㡿 ᇦࢆ⏝࠸ࡿࠋPReF ᢏ⾡ࡣࠊᩘⓒ MPa ௨ୖࡢ㧗ᅽࡀᚲせ࡞㞧ᚤ⏕≀ࡢ⁛⳦ 17 (sterilization)ࢆࢱ࣮ࢤࢵࢺࡍࡿࡢ࡛ࡣ࡞ࡃࠊせ࡞Ⓨ㓝ᚤ⏕≀࡛࠶ࡿ㓝ẕࡢẅ⳦ (pasteurization)࣭άᛶࢆࡍࡿࡇࡼࡾࠊ㐣Ⓨ㓝ࢆไᚚࡍࡿࡇࢆ┠ⓗࡍ ࡿࣉ࣮ࣟࢳ࡛࠶ࡿࠋங㓟⳦ࡣ⮬㌟ࡢ⏕⏘ࡍࡿங㓟ࡼࡿ pH ࡢపୗࡼࡾά ᛶࡍࡿࡓࡵࠊPReF ᢏ⾡࠾࠸࡚ࡣࢱ࣮ࢤࢵࢺࡋ࡞࠸ࠋࡇࢀࡽࡢࡇࡽ PReF ᢏ⾡ࡢ☜❧ࡓࡵࡣࠊ୰㧗ᅽ᮲௳࡛άᛶࡍࡿᅽຊᙅ࠸㓝ẕᰴࡢసฟ ࡀᚲせ࡛࠶ࡿࠋ ➨ 3 㡯 ᅽຊឤཷᛶ㓝ẕࡢసฟ ࡇࢀࡲ࡛ᐇ㦂ᐊࣔࢹࣝ㓝ẕᰴ S. cerevisiae KA31a ᰴࢆぶᰴࡋ࡚ࠊࡑࢀࡼࡾ ࡶ㧗࠸ᅽຊឤཷᛶ⬟ࢆ♧ࡍᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴࢆ⣸እ⥺↷ᑕἲࡼࡿࣛ ࣥࢲ࣒✺↛ኚ␗ᑟධࡼࡾྲྀᚓࡋࡓ(Shigematsu et al., 2010a)ࠋྲྀᚓࡋࡓ a924E1 ᰴࡣࠊKA31a ᰴྠ➼ࡢ࢚ࢱࣀ࣮ࣝⓎ㓝⬟ࢆ᭷ࡋ࡚࠾ࡾࠊᅽຊࡔࡅ࡛࡞ࡃ ᗘ ᑐࡋ࡚ࡶឤཷᛶࢆ♧ࡍࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Shigematsu et al., 2010b)ࠋࡇࢀࡽ ࡢ⾲⌧ᙧ㉁ࢆ᭷ࡍࡿ⏘ᴗ⏝㓝ẕᰴࢆసฟ࡛ࡁࢀࡤࠊPReF ᢏ⾡࠾ࡅࡿ㧗ᅽ༢య ࡢ࠶ࡿ࠸ࡣຍ⇕⤌ࡳྜࢃࡏࡓ㧗ᅽฎ⌮ࡢᛂ⏝᭷┈࡛࠶ࡿࠋࡋࡋ࡞ࡀࡽࠊ a924E1 ᰴࡣࣛࣥࢲ࣒✺↛ኚ␗ࡼࡾྲྀᚓࡉࢀࡓࡓࡵࠊᅽຊឤཷᛶኚ␗ࡀᑟධࡉ ࢀࡓ㑇ఏᏊᗙ࠾ࡼࡧᅽຊឤཷᛶࡢᇶ♏࡞ࡿ࣓࢝ࢽࢬ࣒ࡣࠊᮍࡔ᫂ࡽ࡞ࡗ ࡚࠸࡞࠸ࠋ 18 ➨ 4 ⠇ ◊✲┠ⓗ ᮏ◊✲ࡢ┠ⓗࡣࠊ㧗ᅽ⨨ࡢタഛᢞ㈨ࢥࢫࢺࢆపῶࡍࡿࡓࡵࠊ100㸫200 MPa ⛬ᗘࡢ୰㧗ᅽࡼࡾ㓝ẕ S. cerevisiae ࢆάᛶࡍࡿ PReF ᢏ⾡ࢆసฟࡍࡿࡇ ࡛࠶ࡿࠋ PReF ᢏ⾡ࡼࡾⓎ㓝ᚤ⏕≀ࢆẅ⳦ࡋࡓᚋࡢⓎ㓝㣗ရࡣప ᮲௳࡛ಖᏑ࣭ ὶ㏻ࡉࢀࡿࠋᚑࡗ࡚ࠊPReF ᢏ⾡ࡢᐇ⏝ࡣࠊ༑ศ࡞࢚ࢱࣀ࣮ࣝ⏕⏘⬟ࢆ᭷ࡋࠊ ᑦୟࡘ㧗ᅽឤཷᛶࢆ♧ࡍ⏘ᴗ㓝ẕᰴࡢసฟࡀᚲ㡲࡞ࡿࠋ ࡇࢀࡲ࡛సฟࡋࡓᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴࡣࠊᮍࡔ᫂ࡽ࡞ࡗ࡚࠸࡞ ࠸ᮍ▱ࡢせᅉࡼࡾᅽຊឤཷᛶ⬟ࡀࡉࢀࡓྍ⬟ᛶࡀ࠶ࡿࠋᮏ◊✲࡛ࡣࠊᅽຊ ឤཷᛶࡢ⾲⌧ᆺࢆᘬࡁ㉳ࡇࡍኚ␗㑇ఏᏊ࠾ࡼࡧࡑࡢ࣓࢝ࢽࢬ࣒ࢆ DNA ࣐ࢡ ࣟࣞゎᯒ࠾ࡼࡧ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒࡼࡾゎᯒࡋࡓࠋ 19 ➨ 2 ❶ DNA ࣐ࢡࣟࣞࡼࡿ⥙⨶ⓗ㑇ఏᏊⓎ⌧ゎᯒ ➨ 1 ⠇ ⥴ゝ ➨ 1 㡯 DNA ࣐ࢡࣟࣞゎᯒ DNA ࣐ࢡࣟࣞ(DNA ࢳࢵࣉ)ࡣࠊ1 ᯛᩘ cm2 ࡢࢫࣛࢻࢢࣛࢫ(2.5 cm×8.5 cm)ࡽ࡞ࡿᇶᯈୖࠊᩘⓒࡽᩘ༓✀㢮ࡢ DNA ࣉ࣮ࣟࣈࢆ㓄⨨ࡉࡏࡓ ࡶࡢ࡛࠶ࡿ(ᒾᶫ, 2002)ࠋ┦ྠᛶࡢ㧗࠸㡿ᇦࢆ᭷ࡍࡿ DNA(㑇ఏᏊ)ࡣࠊ୍ᐃࡢ᮲ ௳ୗ࡛ࠊ࠸ゎ㞳⤖ྜࢆ⧞ࡾ㏉ࡍࠋྠ୍ࡢ୍ḟᵓ㐀ࢆᣢࡘ DNA ࡸ┦ྠᛶࡢ 㧗࠸ DNA ࡣ࠸⤖ྜࡀྍ⬟࡛࠶ࡿࡀࠊ୍ḟᵓ㐀ࡀ␗࡞ࡿ DNA ࡣ⤖ྜࡍࡿࡇ ࡣ࡞࠸ࠋᚑࡗ࡚ࠊࢧࣥࣉࣝ୰Ꮡᅾࡍࡿ DNA ࢳࢵࣉୖࡢࣉ࣮ࣟࣈ┦ྠᛶࡢ 㧗࠸ DNA ࡣࠊᩘ༓✀㢮ࡢ DNA ࡢ୰࡛ࡑࡢ┦ྠᛶࡢ㧗࠸ࣉ࣮ࣟࣈࢆ㑅ᢥࡋ࡚⤖ ྜࡍࡿࠋ DNA ࣐ࢡࣟࣞࡣࠊ⣽⬊ࡽᢳฟࡋࡓ mRNA ࢆ⏝࠸࡚ࠊᐇ㦂᮲௳࠾ࡅ ࡿ㑇ఏᏊࡢⓎ⌧㔞ࢆゎᯒࡍࡿࠋࡑࡢࡓࡵ DNA ࣐ࢡࣟࣞゎᯒࡣࠊቑṪࣇ ࢙ࢬࡸ⎔ቃࢫࢺࣞࢫࠊᏛ≀㉁ࠊ✺↛ኚ␗➼ࡢせᅉࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀࡿ㑇 ఏᏊⓎ⌧ࡢ⥙⨶ⓗ࡞ኚࢆ᫂ࡽࡍࡿࡇࡀ࡛ࡁࡿࠋ 20 ➨ 2 㡯 DNA ࣐ࢡࣟࣞゎᯒࡢᛂ⏝ DNA ࣐ࢡࣟࣞゎᯒࡣࠊྠᩘ༓✀㢮௨ୖࡢ㑇ఏᏊࡢⓎ⌧ኚࢆほᐹ ࡍࡿࡇࡀྍ⬟࡛࠶ࡿࠋࡇࢀࡲ࡛࢝ࢻ࣑࣒࢘(Momose and Iwahashi, 2001)ࠊ࢝ ࣉࢧࢩࣥ(Kurita et al., 2002)ࠊ⤖(Odani et al., 2003)ࠊDMSO (Murata et al., 2003)ࠊ ࣛ࢘ࣥࢻࢵࣉ(Sirisattha et al., 2004)ࠊࢺ࢟ࢩࣥ(Iwahashi et al., 2008)ࠊ㖡(Yasokawa et al., 2008)ࠊள㖄(Yasokawa et al., 2010)➼ࡢ㓝ẕࡢᵝࠎ࡞ࢫࢺࣞࢫᛂ⟅㛵ࡍࡿ ሗ࿌ࡀ࠶ࡿࠋFernandes ࡽ(2004)ࡣࠊ200 MPaࠊᐊ ࠊ30 min ࡢ㧗ᅽ᮲௳࡛ฎ⌮ࡋ ࡓᚋࡢ mRNA ࣉࣟࣇࣝࢆሗ࿌ࡋࡓࠋࡋࡋࠊࡇࡢࡼ࠺࡞㧗ᅽ᮲௳࡛ࡣ㌿ ᛂࡣ㐍⾜ࡋ࡞࠸ࡓࡵ(Yayanos and Pollard, 1969)ࠊᙼࡽࡢሗ࿌ࡣ㧗ᅽฎ⌮ᑐࡋ ࡚Ᏻᐃ࡞ mRNA ࡢⓎ⌧ࢆ♧ࡋ࡚࠸ࡿ㐣ࡂ࡞࠸ࠋࡲࡓࠊ200 MPa ⛬ᗘࡢ⮴Ṛⓗ ᮲௳࠾࠸࡚ࡣࠊච㟁Ꮚ㢧ᚤ㙾➼ࡢほᐹ㢗ࡽ࡞ࡅࢀࡤࠊࡑࡢᦆയࢆほᐹࡍࡿ ࡇࡣ㞴ࡋ࠸(Kobori et al., 1995)ࠋ㧗ᅽฎ⌮ࡼࡿ⣽⬊ࡢᙳ㡪ࢆ DNA ࣐ࢡ ࡛ࣟࣞホ౯ࡍࡿࡓࡵࡣࠊ㧗ᅽฎ⌮ࡼࡾ⏕ࡌࡓᦆയࢆᅇࡍࡿኚ ࡍࡿ㑇ఏᏊⓎ⌧ࢆほᐹࡋ࡚ࡑࡢᙳ㡪ࢆ᥎ᐃࡋ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࠋ࠼ࡤࠊ Iwahashi ࡽ(2003)ࡣࠊ4rC ࠾࠸࡚ 180 MPa ࡢ㧗ᅽ᮲௳୍࡛▐ࠊࡲࡓࡣ 40 MPa ࡛ 16 h 㓝ẕ⣽⬊ࢆฎ⌮ࡋࡓࠋࡑࡢᚋ 1 h ᅇᇵ㣴ࡋࡓ⣽⬊ࡽ RNA ࢆᢳฟࡋ࡚ 㑇ఏᏊⓎ⌧ࣉࣟࣇࣝࢆゎᯒࡋࡓࡇࢁࠊ࢜ࣝ࢞ࢿࣛࡸ⭷ᵓ㐀㛵㐃ࡍࡿࢱ ࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢⓎ⌧ࡀࢵࣉࣞࢠ࣮ࣗࣞࢺࡉࢀ࡚࠸ࡓࠋࡇࢀࡽ 21 ࡢ⤖ᯝࡽࠊ⮴Ṛⓗ࡞㧗ᅽ᮲௳᭚ࡉࢀࡓ⣽⬊ࡣࠊ࢜ࣝ࢞ࢿࣛࡸ⭷ᵓ㐀➼ࡢಟ ࢆάⓎ⾜ࡗ࡚࠸ࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࠊ㧗ᅽฎ⌮ࡣ࢜ࣝ࢞ࢿࣛࡸ⭷ᵓ㐀➼ᦆ യࢆᘬࡁ㉳ࡇࡍࡇࡀ♧၀ࡉࢀࡓࠋྠᵝ㠀⮴Ṛⓗ࡞ᅽຊࡢ㐺ᛂࢆゎᯒࡍࡿ ࡓࡵࠊ㓝ẕࡢᑐᩘቑṪᮇ⣽⬊ᑐࡋ࡚ 30 MPaࠊ25°C ࡢᅽຊ᮲௳࡛ฎ⌮ࡋࡓ (Iwahashi et al., 2005)ࠋࡑࡢ⤖ᯝࠊ㠀⮴Ṛⓗᅽຊ᮲௳࠾࠸࡚ࡣࠊࢱࣥࣃࢡ㉁࠾ࡼ ࡧ⭷௦ㅰ㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢⓎ⌧ࡀࢵࣉࣞࢠ࣮ࣗࣞ ࢺࡉࢀ࡚࠸ࡓࠋࡇࢀࡽࡢሗ࿌ࡣࠊDNA ࣐ࢡࣟࣞࡼࡾᅽຊάᛶ࠶ࡿ ࠸ࡣ㧗ᅽ᮲௳ࡢ㐺ᛂ࠸࠺」㞧࡞ࣉࣟࢭࢫࡀゎᯒ࡛ࡁࡿࡇࢆ♧ࡋ࡚࠸ࡿࠋ ➨ 3 㡯 DNA ࣐ࢡࣟࣞゎᯒ࠾ࡅࡿࢡࣟࢫࣁࣈࣜࢲࢮ࣮ࢩࣙࣥ DNA ࣐ࢡࣟࣞゎᯒࡣࠊࠕ࠶ࡿ㑇ఏᏊࡣ┦ྠᛶࡢ㧗࠸㑇ఏᏊࣁࣈࣜ ࢲࢬ(㞧ࠊ⤖ྜ)ࡍࡿࠖ(ᒾᶫ, 2002)࠸࠺ཎ⌮ᇶ࡙ࡃࠋᩘ༓✀㢮ࡶࡢ㑇ఏᏊ Ⓨ⌧ࢆྠ⥙⨶ⓗゎᯒ࡛ࡁࡿⅬࡀ࠶ࡿࡀࠊ┦ྠᛶࡀ㧗࠸ࡀ୍ḟᵓ㐀ࡢ␗ ࡞ࡿ㑇ఏᏊࢆࡶㄗࡗ࡚⤖ྜࡋ࡚ࡋࡲ࠺ࢡࣟࢫࣁࣈࣜࢲࢮ࣮ࢩࣙࣥࢆᘬࡁ㉳ ࡇࡍ(Iwahashi et al., 2007)ࠋ㑇ఏᏊⓎ⌧ࡢ⥙⨶ⓗゎᯒ࠾࠸࡚☜ᐇᛶࢆᘬࡁ㉳ ࡇࡍࢡࣟࢫࣁࣈࣜࢲࢮ࣮ࢩࣙࣥࡣࠊDNA ࢳࢵࣉୖࡢࢃࡎᩘ༑ሷᇶࡢࣉࣟ ࣮ࣈࡼࡗ࡚ᩘ༓✀㢮ࡢ㑇ఏᏊࢆ㆑ูࡍࡿࡓࡵᘬࡁ㉳ࡇࡉࢀࡿࣜࢫࢡ࡛࠶ࡿࠋ ࡋࡋࠊ㑇ఏᏊⓎ⌧ࣉࣟࣇࣝయࢆಠ▔ࡋࠊ᭷ពⓎ⌧ࡀኚືࡍࡿ㑇ఏᏊ⩌ 22 ࡢᶵ⬟ࢆホ౯ࡍࡿࡓࡵࡣࠊ᭷⏝࡛࠶ࡿࠋᚑࡗ࡚ࠊDNA ࣐ࢡࣟࣞゎᯒࡣࠊ Ⓨ⌧ࡀኚࡋࡓ 1 㑇ఏᏊࡢࡳὀ┠ࡍࡿࡢ࡛ࡣ࡞ࡃࠊⓎ⌧ࡢኚࡋࡓ㑇ఏᏊ⩌ ࡢᶵ⬟╔┠ࡍࡿࡁ࠶ࡿࠋࡲࡓࠊࡑࢀࡽࡢ㑇ఏᏊ⩌ࡢⓎ⌧ࡣࠊᚲせᛂࡌ࡚ࢡ ࣟࢫࣁࣈࣜࢲࢮ࣮ࢩࣙࣥࡀᘬࡁ㉳ࡇࡉࢀ࡞࠸ࡼ࠺ཝᐦタィࡉࢀࡓࣉࣛ ࣐࣮ࢆ⏝࠸ࡓ㏫㌿ PCR (RT-PCR)➼ࡼࡗ࡚☜ㄆࡍࡿࡇࡀ㔜せ࡛࠶ࡿࠋ ➨ 4 㡯 ᐇ㦂┠ⓗ ᮏ❶࡛ࡣࠊᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴ࠾ࡼࡧࡑࡢぶᰴ KA31a ᰴࡢ㑇ఏᏊⓎ ⌧ࣉࣟࣇࣝࢆ DNA ࣐ࢡࣟࣞࡼࡗ࡚ẚ㍑ࡍࡿࡇࡼࡾࠊᅽຊឤཷ ᛶ⬟ࢆࡍࡿ㑇ఏⓗせᅉࢆゎᯒࡋࡓࠋDNA ࣐ࢡࣟࣞࡣࠊࢡࣟࢫࣁࣈ ࣜࢲࢮ࣮ࢩࣙࣥࡼࡗ࡚ኚ␗㑇ఏᏊࡢྠᐃ☜ᐇᛶࢆᘬࡁ㉳ࡇࡍࡀࠊ㑇ఏ ᏊⓎ⌧ࣉࣟࣇࣝࡢ᭷ព࡞ኚືࡣኚ␗㑇ఏᏊࡢᶵ⬟ࢆホ౯ࡍࡿࡓࡵᙺ❧ࡘࠋ 23 ➨ 2 ⠇ ᐇ㦂ᮦᩱ࠾ࡼࡧᐇ㦂᪉ἲ ➨ 1 㡯 ⏝⳦ᰴ ᮏᐇ㦂ࡣࠊᐇ㦂ᐊ㓝ẕᰴ S. cerevisiae KA31a ᰴ࠾ࡼࡧᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴࡢᐃᖖᮇ⣽⬊ࢆ⏝ࡋࡓ(⾲ 1)ࠋᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴࡣࠊ⣸እ ⥺↷ᑕࡼࡿ✺↛ኚ␗ࡼࡾྲྀᚓࡋࡓ(Shigematsu et al., 2010a)ࠋࡇࢀࡽࡢ㓝ẕᰴ ࡣࠊ᪂₲⸆⛉Ꮫ 㔜ᯇ ᩍᤵࡼࡾศࡋ࡚࠸ࡓࡔ࠸ࡓࠋ ➨ 2 㡯 ᇵ㣴᮲௳ YPD ᇵᆅ(2.0% peptoneࠊ1.0% yeast extract; Becton Dickinson and Co., NJ, USAࠊ 2.0% glucose; ග⣧⸆ᕤᴗ, 㜰, ᪥ᮏ)ࢆ⏝࠸࡚ࠊ30°Cࠊ48 h ࡢ᮲௳࡛┞ᇵ 㣴ࡋࡓᐃᖖᮇ⣽⬊ࢆᐇ㦂౪ࡋࡓࠋቑṪ᭤⥺ࡣ Hasegawa ࡽ(2012)ࡢ᪉ἲᚑ࠸ࠊ Bio Microplate Reader HiTSTM (Scinics, Ⲉᇛ, ᪥ᮏ)ࢆ⏝࠸࡚ 30°Cࠊ120 h ࡢ᮲௳࡛ ᐃࡋࡓࠋ ➨ 3 㡯 RNA ᢳฟ᪉ἲ YPD ᇵᆅ࡛ᇵ㣴ࡋࡓ㓝ẕ⣽⬊ࡣ 15,000 rpmࠊ4°Cࠊ1 min ࡢ᮲௳࡛㐲ᚰศ㞳(MX301; Tomy Seiko, ᮾி, ᪥ᮏ)ࡼࡾᅇࡋࡓࠋTotal RNA ࡣ Fast RNA® Pro Red 24 Kit (MP Biomedicals, CA, USA)ࢆ⏝࠸࡚ࣇ࢙࣮ࣀ࣮ࣝ-ࢡ࣒ࣟࣟ࣍ࣝἲࡼࡾᢳฟ ࡋࡓࠋ⣽⬊◚○ࢆ Multi-Beads Shocker® (ᏳჾᲔ, 㜰, ᪥ᮏ)࡛ 10 min ࠊࢡ ࣒ࣟࣟ࣍ࣝฎ⌮ࢆ 2 ᅇኚ᭦ࡋࡓ௨እࡣᢳฟ࢟ࢵࢺࡢࣉࣟࢺࢥࣝᚑࡗࡓࠋRNA ⃰ᗘ࠾ࡼࡧ⣧ᗘࡣ 260 nm ࡢ྾ගᗘ࠾ࡼࡧ Agilent 2100 BioanalyzerTM (Agilent Technologies, CA, USA)ࡼࡾ ᐃࡋࡓࠋ ➨ 4 㡯 DNA ࣐ࢡࣟࣞゎᯒ᪉ἲ DNA ࣐ࢡࣟࣞࡼࡿ㑇ఏᏊⓎ⌧ࡢ⥙⨶ⓗゎᯒࡣࠊYeast Oligo Microarray Kit (V2)TM (Agilent Technologies)ࢆ⏝࠸࡚ゎᯒࡋࡓࠋࡇࡢ࣐ࢡ࡛ࣟࣞࡣࠊS. cerevisiae S288c ᰴࡢ 6,256 㑇ఏᏊᑐᛂࡍࡿ DNA ࣉ࣮ࣟࣈࢆ⏝࠸ࡓࠋ㑇ఏᏊⓎ ⌧ゎᯒࡣ 1 ⰍἲࡼࡾゎᯒࡋࡓࠋcDNA ࡣ Quick Amp Labeling KitTM (Agilent Technologies)ࢆ⏝࠸࡚ total RNA ࡽㄪ〇ࡋࡓࠋcRNA ࡣࠊCy5 Ⰽ⣲ࡼࡾ T7 RNA polymerase (Agilent Technologies)ࢆ⏝࠸࡚ࣛ࣋ࣝࡋࡓࠋcDNA ࠾ࡼࡧ cRNAࠊ Cy5 ࡼࡿࣛ࣋ࣝࠊࣉ࣮ࣟࣈࡢࣁࣈࣜࢲࢮ࣮ࢩࣙࣥࡣ Ecogenomics (⚟ ᒸ, ᪥ᮏ)ጤクࡋࡓࠋ 25 ➨ 5 㡯 㑇ఏᏊⓎ⌧ࡢศ㢮ゎᯒ᪉ἲ ࡑࢀࡒࢀࡢ ORF(Open Reading Frame)ࡽ᳨ฟࡉࢀࡓࢩࢢࢼࣝࡣࠊࢡ࢜ࣥࢱ ࣝἲࡼࡾṇつࡋࡓࠋKA31a ᰴ᳨࡛ฟࡉࢀࡓࢩࢢࢼࣝẚ࡚ 2 ಸ௨ୖ࠶ࡿ ࠸ࡣ༙ศ௨ୗ a924E1 ᰴࡢࢩࢢࢼࣝᙉᗘࡀኚࡋࡓ㑇ఏᏊࢆࡑࢀࡒࢀࢵࣉࣞ ࢠ࣮ࣗࣞࢺ㑇ఏᏊࠊࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊࡋࡓࠋྛ㑇ఏᏊࡣࢫࢳ࣮ࣗࢹࣥ ࢺࡢ t ᳨ᐃ(p < 0.05)ࡼࡾ⤫ィฎ⌮ࡋࡓࠋⓎ⌧ࡀኚࡋࡓ㑇ఏᏊࡣ MIPS GenRE CYGD database (http://mips.helmholtz-muenchen.de/genre/proj/yeast/)ࡼࡾゎᯒࡋ ࡓࠋᮏ◊✲ࡢ DNA ࣐ࢡࣟࣞࢹ࣮ࢱࡣ MIAME ᇶ‽‽ᣐࡋࠊ⏕ࢹ࣮ࢱࡣ Gene Expression Omnibus (GEO) database ᐤクࡋࡓ(GSE55120)ࠋ ➨ 6 㡯 quantitative PCR ᪉ἲ ୖグࡢ᪉ἲ࡛㓝ẕ⣽⬊ࡽᢳฟࡉࢀࡓ total RNA ࡣࠊReverTra AceR qPCR RT Master Mix (TOYOBO, Osaka, Japan)ࢆ⏝࠸࡚ 37°Cࠊ15 min ࡢ᮲௳࡛㏫㌿ࡋࡓࠋ DNA ࣐ࢡࣟࣞࡢ⤖ᯝᇶ࡙ࡁࠊࢵࣉࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊ࠾ࡼࡧࢲ࢘ࣥ ࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊࡢࣉ࣐࣮ࣛࢆタィ(⾲ 2)ࡋࠊPower SYBR® Green Master Mix (Applied Biosystems, CA, USA) ࢆ ⏝ ࠸ ࡚ StepOnePlusTM rial-time PCR System (Applied Biosystems)ࡼࡾቑᖜࡋࡓ(95°C for 10 min, and 40 cycles: denaturation at 95°C for 15 s, annealing and extension at 60°C for 2 min)ࠋᚓࡽࢀࡓ Ct ್ࡽ┦ᑐⓎ 26 ⌧ࣞ࣋ࣝࢆ⟬ฟࡋࡓࠋCDC48 㑇ఏᏊࡣ⣽⬊࿘ᮇ㛵㐃ࡍࡿᚲ㡲㑇ఏᏊ࡛࠶ࡾࠊ a924E1 ᰴ࠾ࡼࡧ KA31a ᰴࡢ୧ᰴ࠾࠸࡚ྠ➼ࡢⓎ⌧㔞࡛࠶ࡗࡓࡓࡵࣁ࢘ࢫ ࣮࢟ࣆࣥࢢ㑇ఏᏊࡋ࡚⏝ࡋࡓࠋ 27 ➨ 3 ⠇ ⤖ᯝ ➨ 1 㡯 㑇ఏᏊⓎࣉࣟࣇࣝࡢᴫせ ᮏ❶࡛ࡣࠊDNA ࣐ࢡࣟࣞࡼࡾᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴࡢ㑇ఏᏊ Ⓨ⌧ࣉࣟࣇࣝࢆゎᯒࡋࡓࠋTotal RNA ࡣ a924E1 ᰴ࠾ࡼࡧ KA31a ᰴࡽࡑࢀ ࡒࢀ 4 ᅇࡢ⊂❧ࡋࡓᐇ㦂ࡼࡾᢳฟࡉࢀࡓࠋᢳฟ RNA ࡢ⃰ᗘࡣ࠾ࡼࡑ 1.2 μg/μL ࡛࠶ࡿࠋࡇࡢ RNA ࢆ௨ୗࡢ DNA ࣐ࢡࣟࣞ࠾ࡼࡧ RT-PCR ౪ࡋࡓࠋ DNA ࣐ࢡࣟࣞࡼࡾ᳨ฟࡉࢀࡓࢩࢢࢼࣝࢹ࣮ࢱࡣࢡ࢜ࣥࢱࣝἲࡼ ࡾṇつࡋࠊ5,821 㑇ఏᏊࡢⓎ⌧ࢹ࣮ࢱ 8 ࢭࢵࢺࢆྲྀᚓࡋࡓࠋࡇࢀࡽࡢ㑇ఏᏊࡢ ࠺ࡕࠊa924E1 ᰴࡢ 498 㑇ఏᏊࡢⓎ⌧ࣞ࣋ࣝࡣ KA31a ᰴࡢ㑇ఏᏊࡢⓎ⌧ࣞ࣋ࣝࡼ ࡾࡶ᭷ព㧗ࡃ(p < 0.05)ࠊ649 㑇ఏᏊࡢⓎ⌧ࣞ࣋ࣝࡣ᭷ពపࡗࡓ(p < 0.05)ࠋ ➨ 2 㡯 ࢵࣉࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊⓎ⌧ࡢゎᯒ a924E1 ᰴ࠾࠸࡚ࢵࣉࣞࢠ࣮ࣗࣞࢺࡉࢀࡓ 498 㑇ఏᏊࡣࠊMIPS GenRE CYGD database ࡼࡾศ㢮ゎᯒࡉࢀࡓࠋⓎ⌧ࡢኚືࡋࡓ㑇ఏᏊࡢྜࡀ᭱ࡶ㧗ࡃࠊ p ್ࡀ᭱ࡶప࠸ᶵ⬟࢝ࢸࢦ࣮ࣜࡣ“Energy”࡛࠶ࡗࡓ(⾲ 3)ࠋ“Energy”࢝ࢸࢦ࣮ࣜ ศ㢮ࡉࢀࡿ 367 㑇ఏᏊࡢ࠺ࡕࢩࢺࢡ࣒ࣟ c 㓟㓝⣲ࠊATP ྜᡂ㓝⣲ࠊcoenzyme Q ➼㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊ(COX1ࠊAI1ࠊCOX17ࠊCOX18ࠊ 28 CYC7ࠊPET191ࠊSHY1ࠊNCA2ࠊCOQ8 㑇ఏᏊ)ࡸ࣑ࢺࢥࣥࢻࣜࡢࣜ࣎ࢯ࣮࣒ྜ ᡂ㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊ➼ 45 㑇ఏᏊ(MRPL1ࠊMRPS35ࠊ MBA1 㑇ఏᏊ)ࡀࢵࣉࣞࢠ࣮ࣗࣞࢺࡉࢀ࡚࠸ࡓࠋ “Biogenesis of cellular components”࢝ࢸࢦ࣮ࣜࡣࢵࣉࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊࡢ ྜࡀ 2 ␒┠ከࡗࡓ(⾲ 3)ࠋྠ࢝ࢸࢦ࣮ࣜศ㢮ࡉࢀࡿ 862 㑇ఏᏊࡢ࠺ࡕ 88 㑇ఏᏊࡀࢵࣉࣞࢠ࣮ࣗࣞࢺࡉࢀ࡚࠸ࡓࠋࡲࡓࠊྠ࢝ࢸࢦ࣮ࣜࡢୗ࢝ࢸࢦ࣮ࣜ ࡛࠶ࡿ“Mitochondrion”ࢧࣈ࢝ࢸࢦ࣮ࣜศ㢮ࡉࢀࡿ 171 㑇ఏᏊࡢ࠺ࡕ 32 㑇ఏᏊ ࡀࢵࣉࣞࢠ࣮ࣗࣞࢺࡉࢀ࡚࠸ࡓࠋࡑࢀࡽࡢࢵࣉࣞࢠ࣮ࣗࣞࢺࡉࢀࡓ㑇ఏᏊ ࡢ࠺ࡕࠊ21 㑇ఏᏊࡀ࣑ࢺࢥࣥࢻࣜࣜ࣎ࢯ࣮࣒㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻ ࡍࡿ㑇ఏᏊ(MRP51ࠊMRPL1ࠊMRPS35ࠊRSM25ࠊVAR1 㑇ఏᏊ)࡛࠶ࡗࡓࠋ ࡑࢀࡒࢀࡢ㑇ఏᏊࡀࢥ࣮ࢻࡍࡿࢱࣥࣃࢡ㉁ࡢᒁᅾ⨨ศ㢮(⾲ 4)࠾࠸࡚ࡣࠊ ࣑ࢺࢥࣥࢻࣜᒁᅾࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ“Mitochondria”࢝ࢸࢦ࣮ࣜ ศ㢮ࡉࢀࡿ㑇ఏᏊ⩌ࡀ᭱ࡶప࠸ p ್ࢆ♧ࡋࡓ(p < 0.01)ࠋࡇࢀࡽࡢࢵࣉࣞࢠࣗ ࣮ࣞࢺࡉࢀࡓ㑇ఏᏊࡀࢥ࣮ࢻࡋ࡚࠸ࡿࢱࣥࣃࢡ㉁ࡣ㓟ⓗࣜࣥ㓟㛵ࡋ࡚ ࠾ࡾࠊ࢚ࢿࣝࢠ࣮⏘⏕㔜せ࡛࠶ࡿࠋࡇࢀࡽࡢ⤖ᯝࡣࠊa924E1 ᰴ࠾࠸࡚ᅽຊ ឤཷᛶኚ␗ᑟධࡼࡾ࣑ࢺࢥࣥࢻࣜᶵ⬟㛵㐃ࡍࡿ㑇ఏᏊᙳ㡪ࡀ⾲ࢀ࡚࠸ ࡿࡇࢆ♧၀ࡋ࡚࠸ࡿࠋ 29 ➨ 3 㡯 ࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊⓎ⌧ࡢゎᯒ a924E1 ᰴ࠾࠸࡚ࠊࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺࡋࡓ㑇ఏᏊࡣ 649 㑇ఏᏊ᳨ฟࡉࢀࡓ (⾲ 5)ࠋࡇࢀࡽࡢ㑇ఏᏊࡣ MIPS GenRE CYGD database ࡼࡾศ㢮ࡉࢀࠊ“Protein synthesis”ࠊ“Transcription”ࠊ“Binding protein”࢝ࢸࢦ࣮ࣜࡢ 3 ࢝ࢸࢦ࣮ࣜࡀྛ࢝ࢸ ࢦ࣮ࣜศ㢮ࡉࢀࡿ㑇ఏᏊ࠾࠸࡚ኚື㑇ఏᏊࡢ㧗࠸ྜࠊప࠸ p ್ࢆ♧ࡋࡓ (⾲ 5)ࠋ“Protein synthesis”࢝ࢸࢦ࣮ࣜศ㢮ࡉࢀࡿ㑇ఏᏊࡢ࠺ࡕ 155 㑇ఏᏊࡀࢲ ࢘ࣥࣞࢠ࣮ࣗࣞࢺࡉࢀࠊ“Transcription”࢝ࢸࢦ࣮࡛ࣜࡣ 1,077 㑇ఏᏊࡢ࠺ࡕ 155 㑇 ఏᏊࠊ“Binding protein”࢝ࢸࢦ࣮࡛ࣜࡣ 1,049 㑇ఏᏊࡢ࠺ࡕ 148 㑇ఏᏊࡀࢲ࢘ࣥ ࣞࢠ࣮ࣗࣞࢺࡋࡓࠋRPS3ࠊRPS5ࠊRPS31ࠊRPL10ࠊRPL30 㑇ఏᏊ➼ࡢࡇࢀࡽࡢ࢝ ࢸࢦ࣮ࣜศ㢮ࡉࢀࡓࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊࡢ࠸ࡃࡘࡣࠊࣜ࣎ࢯ࣮࣒ࡢ ⏕ྜᡂࡸ rRNA ࡢྜᡂࠊࣉࣟࢭࢵࢩࣥࢢࠊಟ㣭➼㛵㐃ࡍࡿࠋࡇࢀࡽࡢ㑇ఏᏊࡀ ࢥ࣮ࢻࡍࡿࢱࣥࣃࢡ㉁ࡣ“Cytoplasm”࠾ࡼࡧ“Nucleus”ᒁᅾࡋ࡚࠸ࡓ(⾲ 6)ࠋ ࡲࡓࠊࣂࣜࣥࠊࣟࢩࣥࠊࢯࣟࢩࣥࠊࣝࢠࢽࣥࠊࢭࣜࣥࠊࢺࣜࣉࢺࣇࣥ ➼ࡢ࣑ࣀ㓟ࡢ⏕ྜᡂ㛵㐃ࡍࡿࢧࣈ࢝ࢸࢦ࣮ࣜศ㢮ࡉࢀࡓ㑇ఏᏊࡶࢲ࢘ࣥ ࣞࢠ࣮ࣗࣞࢺࡋࡓࠋࡇࢀࡽࡢ⤖ᯝࡣࠊኚ␗ᰴ࠾࠸࡚ࢱࣥࣃࢡ㉁࠾ࡼࡧ࣑ࣀ㓟 ࡢ⏕ྜᡂ㛵ࡍࡿάᛶࡀῶᑡࡋ࡚࠸ࡿࡇࢆ♧၀ࡋ࡚࠸ࡿࠋ 30 ➨ 4 㡯 quantitative PCR ࡼࡿ㑇ఏᏊⓎ⌧ゎᯒࡢホ౯ ࢵࣉࣞࢠ࣮ࣗࣞࢺ࠾ࡼࡧࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊࡢⓎ⌧ࢆ☜ㄆࡍࡿࡓࡵ ྛ㑇ఏᏊᑐᛂࡍࡿࣉ࣐࣮ࣛࢆタィࡋࡓ(⾲ 2)ࠋquantitative PCR ࡼࡾ a924E1 ᰴ࠾ࡼࡧ KA31a ᰴࡢ㑇ఏᏊⓎ⌧ࢆホ౯ࡋࡓ(⾲ 7)ࠋa924E1 ᰴࡢࢵࣉࣞ ࢠ࣮ࣗࣞࢺࡋࡓ㑇ఏᏊࡢ┦ᑐⓎ⌧ࣞ࣋ࣝࡣࠊKA31a ᰴࡢ┦ᑐⓎ⌧ࣞ࣋ࣝࡼࡾࡶ 2 ಸ௨ୖ㧗ࡗࡓࠋ୍᪉ࠊa924E1 ᰴࡢࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊࡢ┦ᑐⓎ⌧ࣞ ࣋ࣝࡣ KA31a ᰴẚ㍑ࡋ࡚༙ศ௨ୗ࡛࠶ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝࡣࠊDNA ࣐ࢡࣟ ࣞࡼࡿ㑇ఏᏊⓎ⌧ゎᯒ⤖ᯝࡢጇᙜᛶࢆᨭᣢࡋ࡚࠸ࡿࠋࡋࡋ࡞ࡀࡽࠊDNA ࣐ࢡࣟࣞࡼࡗ࡚ࢵࣉࣞࢠ࣮ࣗࣞࢺࡉࢀ࡚࠸ࡓ a924E1 ᰴࡢ COX1 㑇ఏ Ꮚࡢ┦ᑐⓎ⌧ࣞ࣋ࣝࡣ࡚ࡶᑡ࡞ࡗࡓࠋCOX1 㑇ఏᏊࡢูࡢ㡿ᇦࢆࢱ࣮ࢤࢵࢺ ࡋࡓࣉ࣐࣮࡛ࣛ☜ㄆࡋࡓࡀࠊⓎ⌧ࢆほᐹࡍࡿࡇࡣ࡛ࡁ࡞ࡗࡓ(⾲ 7)ࠋ 31 ➨ 4 ⠇ ⪃ᐹ ᮏ❶࡛ࡣࠊᅽຊឤཷᛶ⬟ࡢᶵᵓࢆ᫂ࡽࡍࡿࡓࡵࠊDNA ࣐ࢡࣟ ࣞࡼࡾᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴࡢ㑇ఏᏊⓎ⌧ࣉࣟࣇࣝࢆࡑࡢぶᰴ KA31a ᰴࡢࡶࡢẚ㍑ࡋࡓࠋࡑࡢ⤖ᯝࠊa924E1 ᰴࡢ 1,000 ௨ୖࡢ㑇ఏᏊࡢⓎ⌧ ࡀ᭷ពኚࡋࡓࡇࡀ᫂ࡽ࡞ࡗࡓ(p < 0.05)ࠋ≉࢚ࢿࣝࢠ࣮௦ㅰᶵ⬟ 㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢⓎ⌧ࡀࢵࣉࣞࢠ࣮ࣗࣞࢺࡋ(⾲ 3)ࠊ ࣑ࣀ㓟ࡸࢱࣥࣃࢡ㉁ࡢ⏕ྜᡂ㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢⓎ ⌧ࡀࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺࡋࡓ(⾲ 5)ࠋࢵࣉࣞࢠ࣮ࣗࣞࢺࡋࡓ㑇ఏᏊࡢከࡃࡣ࣑ ࢺࢥࣥࢻࣜᒁᅾࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊ࡛࠶ࡗࡓࠋDNA ࣐ࢡ ࣟࣞࡼࡿ㑇ఏᏊⓎ⌧ࡢゎᯒ࠾࠸࡚ὀពࡋ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࡇࡣࠊⓎ ⌧ࡀኚືࡋࡓᶵ⬟㑇ఏᏊ⩌ࡀࠊ࡞ࡐⓎ⌧ࢆኚືࡉࡏࡿᚲせࡀ࠶ࡗࡓࡢ DNA ࣐ ࢡࣟࣞゎᯒࡢ⤖ᯝయࢆಠ▔ⓗ⪃ᐹࡍࡿࡇ࡛࠶ࡿࠋ࣑ࢺࢥࣥࢻࣜ 㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡀࢵࣉࣞࢠ࣮ࣗࣞࢺࡉࢀࡓࡇ ࡽࠊa924E1 ᰴ࠾࠸࡚࣑ࢺࢥࣥࢻࣜᶵ⬟ࡀ㔜せ࡛࠶ࡿࡇࡀ⪃࠼ࡽࢀࡿࠋࡑ ࡢཎᅉࡋ࡚ࡣࠊᅽຊឤཷᛶኚ␗ࡢᑟධࡼࡾձ࣑ࢺࢥࣥࢻࣜࡀศゎ࠶ࡿ࠸ ࡣᶵ⬟࡞ࡗࡓࡢࠊղ࣑ࢺࢥࣥࢻࣜᶵ⬟ࡀᙉࡉࢀࡓࡢࢃࡽ࡞࠸ࠋ ࡇࡢ⤖ᯝేࡏ࡚ࠊࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺࡋࡓ㑇ఏᏊ⩌ࡀ࣑ࣀ㓟ࡸࢱࣥࣃࢡ㉁ 32 ࡢ⏕ྜᡂ㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡋ࡚࠸ࡓࡇࡽࠊᅽຊឤཷᛶ⬟ࡣ a924E1 ᰴࡢ࣑ࢺࢥࣥࢻࣜᶵ⬟ࡀపୗࡍࡿࡼ࠺࡞ᙳ㡪ࢆᘬࡁ㉳ࡇࡋ࡚࠸ࡿྍ⬟ ᛶࡀ⪃࠼ࡽࢀࡿࠋ RT-PCR ࡼࡾ㑇ఏᏊⓎ⌧ࢆホ౯ࡋࡓ⤖ᯝࠊa924E1 ᰴࡢࢵࣉࣞࢠ࣮ࣗࣞࢺ ࡋࡓ㑇ఏᏊࡢ┦ᑐⓎ⌧ࣞ࣋ࣝࡣࠊKA31a ᰴࡢ┦ᑐⓎ⌧ࣞ࣋ࣝࡼࡾࡶ 2 ಸ௨ୖ㧗 ࠸್ࢆ♧ࡋࡓ(⾲ 7)ࠋࡋࡋ࡞ࡀࡽࠊDNA ࣐ࢡࣟࣞゎᯒࡼࡾࢵࣉࣞࢠ ࣮ࣗࣞࢺࡋ࡚࠸ࡓ࣑ࢺࢥࣥࢻࣜࡢ㓟ⓗࣜࣥ㓟㛵㐃ࡍࡿࢩࢺࢡ࣒ࣟ c 㓟 㓝⣲ࢆࢥ࣮ࢻࡍࡿ COX1 㑇ఏᏊࡢⓎ⌧ࡣ☜ㄆ࡛ࡁ࡞ࡗࡓ(⾲ 7)ࠋ DNA ࣐ࢡࣟࣞࡣ DNA ࢳࢵࣉୖࡢࢃࡎᩘ༑ሷᇶࡢࣉ࣮ࣟࣈࡼࡗ࡚ ᩘ༓ࡢ㑇ఏᏊࢆ㆑ูࡍࡿ᪉ἲ࡛࠶ࡿࠋࢡࣟࢫࣁࣈࣜࢲࢮ࣮ࢩࣙࣥࡍࡿࡇ ࡀ↓࠸ࡼ࠺ཝᐦタィࡉࢀࡓ PCR ⏝ࡢࣉ࣐࣮ࣛ␗࡞ࡾࠊDNA ࣐ࢡࣟ ࣞࡢࣉ࣮ࣟࣈࡣࢡࣟࢫࣁࣈࣜࢲࢮ࣮ࢩࣙࣥࡍࡿࣜࢫࢡࡼࡾࡶᩘ༓✀㢮௨ ୖࡢ㑇ఏᏊࡢⓎ⌧ࢆ⥙⨶ⓗゎᯒࡍࡿࡇࢆඃඛࡋ࡚࠸ࡿࠋࡑࡢࡓࡵࠊࡇࡢぢ ࡅୖࡢ COX1 㑇ఏᏊࡢࢵࣉࣞࢠ࣮ࣗࣞࢩࣙࣥࡣࠊDNA ࣐ࢡࣟࣞ≉ ᭷ࡢࢡࣟࢫࣁࣈࣜࢲࢮ࣮ࢩ࡛ࣙࣥ࠶ࡿ⪃࠼ࡽࢀࡿࠋDNA ࣉ࣮ࣟࣈࡢሷᇶ 㓄ิࡢ┦ྠᛶࡸ⺯ගᙉᗘࡢࡤࡽࡘࡁࠊ⣽⬊ࡢᇵ㣴᮲௳➼ࡀཎᅉ࡛ࡋࡤࡋࡤࢡࣟ ࢫࣁࣈࣜࢲࢮ࣮ࢩࣙࣥࡀᘬࡁ㉳ࡇࡉࢀࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Iwahashi et al., 2007)ࠋᐇ㝿 DNA ࢳࢵࣉୖࡢ COX1 㑇ఏᏊࡢࣉ࣮ࣟࣈࡣࠊࡢ㑇ఏᏊᴟ 33 ࡵ࡚㧗࠸┦ྠᛶࢆ♧ࡋࡓࠋ࠼ࡤࠊCOX1 㑇ఏᏊࣉ࣮ࣟࣈࡢ 40-60 bp 㒊ศࡣࠊ5࣍ࢫ࣍ࣜ࣎ࢩࣝ-1-α-ࣆࣟࣜࣥ㓟ྜᡂ㓝⣲ࢆࢥ࣮ࢻࡍࡿ PRS2 㑇ఏᏊࡢሷᇶ㓄ิ ୍⮴ࡋࡓࠋຍ࠼࡚ࠊCOX1 㑇ఏᏊࡢⓎ⌧ࡘ࠸࡚ࠊ㑇ఏᏊ㓄ิࡢ␗࡞ࡿ㡿ᇦࢆ ᑐ㇟ࡋࡓ quantitative PCR ࡛ゎᯒࡋࡓࡇࢁࠊⓎ⌧ࡣ☜ㄆ࡛ࡁ࡞ࡗࡓ(⾲ 7)ࠋ ࡇࢀࡽࡢ⤖ᯝࡣࠊa924E1 ᰴ࠾࠸࡚ COX1 㑇ఏᏊࡢⓎ⌧ࡀࢵࣉࣞࢠ࣮ࣗࣞࢺ ࡋ࡚࠸࡞࠸ࡇࢆ♧၀ࡋ࡚࠸ࡿࠋ ᮏ❶ࡢ⤖ᯝࢆࡲࡵࡿࠊa924E1 ᰴࡣᅽຊឤཷᛶኚ␗ࡢᑟධࡼࡾ࣑ࢺࢥࣥ ࢻࣜࡸ࣑ࣀ㓟➼ࡢࢱࣥࣃࢡ㉁ࡢ⏕ྜᡂ㛵㐃ࡍࡿᶵ⬟ᙳ㡪ࡀ⏕ࡌࡓࡇ ࡀ♧၀ࡉࢀࡓࠋ࣑ࢺࢥࣥࢻࣜࡣࠊᵝࠎ࡞࣑ࣀ㓟➼ࡢ୰㛫௦ㅰ⏘≀ࡢ⏕ྜᡂࡢ せ࡞௦ㅰ⤒㊰࡛࠶ࡾࠊ㔜せ࡞ᙺࢆᢸ࠺ჾᐁ࡛࠶ࡿࠋ➨ 3 ❶࡛ࡣࠊDNA ࣐ ࢡࣟࣞゎᯒᇶ࡙ࡁࠊa924E1 ᰴࡢᅽຊឤཷᛶ⬟ࡀཬࡰࡍ࣑ࢺࢥࣥࢻࣜᶵ ⬟ࡢᙳ㡪ࡘ࠸࡚ゎᯒࡍࡿࠋ 34 ➨ 3 ❶ ࣑ࢺࢥࣥࢻࣜᶵ⬟ࡢゎᯒ ➨ 1 ⠇ ⥴ゝ ➨ 1 㡯 㓝ẕ࣑ࢺࢥࣥࢻࣜ ࣑ࢺࢥࣥࢻࣜࡣ㓟ⓗࣜࣥ㓟ࡼࡿ࢚ࢿࣝࢠ࣮⏕⏘ࢆྖࡿ⣽⬊ᑠჾᐁ࡛ ࠶ࡿࠋ࣑ࢺࢥࣥࢻࣜࡣ⊂⮬ DNA(࣑ࢺࢥࣥࢻࣜ DNA)ࢆᣢࡕࠊศࠊቑṪ ࢆ⾜࠺ࡇࡀ࡛ࡁࡿࠋ㓝ẕࡢ࣑ࢺࢥࣥࢻࣜ DNA ࡣ࠾ࡼࡑ 76 kbp ࡛࠶ࡾࠊࣄࢺ ࡣ࠾ࡼࡑ 16.5 kbp ࡛࠶ࡿ(⚟ཎ, 1986)ࠋࢇࡢ┿᰾⏕≀ࡣ࢚ࢿࣝࢠ࣮⋓ᚓࢆ ዲẼ྾౫Ꮡࡋ࡚࠸ࡿࡓࡵࠊ࣑ࢺࢥࣥࢻࣜ DNA ࡢḞᦆ➼㉳ᅉࡍࡿ࣑ࢺ ࢥࣥࢻࣜᶵ⬟ࡢపୗࡣ⮴ⓗ࡛࠶ࡿࠋ࣑ࢺࢥࣥࢻࣜ DNA ࡢḞᦆ㛵㐃ࡋ࡚ ࠸ࡿࣄࢺࡢ㑇ఏࡶᑡ࡞ࡃ࡞࠸ࠋࡲࡓࠊ࣑ࢺࢥࣥࢻࣜࡢ㓟ⓗࣜࣥ㓟ࡼࡾ ⏕ᡂࡉࢀࡿάᛶ㓟⣲ࡀ⒴ࢆᘬࡁ㉳ࡇࡍࡇࡶ♧၀ࡉࢀ࡚࠸ࡿࠋ 㓝ẕࡣ࢚ࢱࣀ࣮ࣝⓎ㓝ࡼࡾ⏕Ꮡࡍࡿࡇࡀྍ⬟࡛࠶ࡿࡓࡵࠊ࣑ࢺࢥࣥࢻࣜ DNA ࡢḞᦆࡣࣄࢺ⮴ⓗ࡛ࡣ࡞࠸ࠋᐇ㝿 Ephrussi ࡽ(1955)ࡶሗ࿌ࡋ࡚ ࠸ࡿࡼ࠺㓝ẕࡢ྾Ḟᦆᰴࡘ࠸࡚ࡣᩘከࡃሗ࿌ࡉࢀ࡚࠾ࡾࠊ≉࣑ࢺࢥࣥ ࢻࣜ DNA ࡀḞኻࡋࡓᰴࢆ rho㸫ᰴࠊḞኻࡋࡓᰴࢆ rho0 ᰴࡪࠋ㓝ẕ ࡣࣄࢺࡢ྾ᶵ⬟㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢ࣍ࣔࣟࢢࢆᩘከ 35 ࡃ᭷ࡋ࡚࠸ࡿࠋࡑࡢࡓࡵ࣑ࢺࢥࣥࢻࣜࡀ㛵㐃ࡍࡿ㑇ఏࡸ⒴ࢆጞࡵࡋࡓ ከࡃࡢẼࡸ྾㜼ᐖ➼ᑐࡍࡿᇶ♏◊✲ࡋ࡚㓝ẕࡢ࣑ࢺࢥࣥࢻࣜ◊✲ ࡀάⓎ㐍ࡵࡽࢀ࡚࠸ࡿ (⚟ཎ, 1986)ࠋ ➨ 2 㡯 ᐇ㦂┠ⓗ ➨ 2 ❶ࡢ DNA ࣐ࢡࣟࣞゎᯒࡼࡾࠊa924E1 ᰴࡢᅽຊឤཷᛶ࣑ࢺࢥ ࣥࢻࣜࡢ㛵㐃ᛶࡀ♧၀ࡉࢀࡓࠋ࣑ࢺࢥࣥࢻࣜࡣ྾ᶵ⬟ࡸ⣽⬊ෆ௦ㅰࡢ୰ 㛫௦ㅰ⏘≀ࢆ⏕⏘ࡍࡿ㔜せ࡞ჾᐁ࡛࠶ࡿࠋᮏ❶࡛ࡣ࣑ࢺࢥࣥࢻࣜᶵ⬟ࢆ⾲⌧ ᙧ㉁ゎᯒࡼࡾホ౯ࡋࡓࠋ 36 ➨ 2 ⠇ ᐇ㦂ᮦᩱ࠾ࡼࡧᐇ㦂᪉ἲ ➨ 1 㡯 ⏝⳦ᰴ ᮏᐇ㦂ࡣࠊᐇ㦂ᐊ㓝ẕᰴ S. cerevisiae KA31a ᰴ࠾ࡼࡧᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴࡢᐃᖖᮇ⣽⬊ࢆ⏝ࡋࡓ(⾲ 1)ࠋࡇࢀࡽࡢ㓝ẕᰴࡣࠊ᪂₲⸆⛉Ꮫ 㔜 ᯇ ᩍᤵࡼࡾศࡋ࡚࠸ࡓࡔ࠸ࡓࠋࡲࡓࠊ➨ 3 㡯࡛సฟࡋࡓಸయᰴࢆ⏝ ࡋࡓ(⾲ 1)ࠋ ➨ 2 㡯 ᇵ㣴᮲௳ YPD ᇵᆅ(2.0% peptoneࠊ1.0% yeast extractࠊ2.0% glucose)ࢆ⏝࠸࡚ࠊ30°Cࠊ48 h ࡢ᮲௳࡛┞ᇵ㣴ࡋࡓᐃᖖᮇ⣽⬊ࢆᐇ㦂౪ࡋࡓࠋቑṪ᭤⥺ࡣ Hasegawa ࡽ (2012)ࡢ᪉ἲᚑ࠸ࠊBio Microplate Reader HiTSTM ࢆ⏝࠸࡚ 30°Cࠊ120 h ࡢ᮲௳ ࡛ ᐃࡋࡓࠋ ➨ 3 㡯 ಸయᰴࡢసฟ ᮏᐇ㦂࡛⏝ࡋࡓಸయᰴࡣࠊᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴ(a ᆺ୍ಸయᰴ) 㔝⏕ᆺᰴ KA31α ᰴ(α ᆺ୍ಸయᰴ)ࡢ᥋ྜࡼࡾసฟࡋࡓࠋ୧ᰴࡢᇵ㣴ᾮ 300 μL ࢆ YPD ᇵᆅ 2.4 mL ຍ࠼࡚ࡼࡃΰྜࡋࠊ30°Cࠊ24 h ࡢ᮲௳࡛㟼⨨ᇵ㣴ࡋࡓࠋᇵ 37 㣴ᚋࠊ┦ᕪ㢧ᚤ㙾ࡼࡾ᥋ྜᏊࢆ☜ㄆࡋ࡚ YPD ᐮኳᇵᆅୖ᥋ྜ⣽⬊ࢆ ࢯ࣮ࣞࢩࣙࣥࡋࠊ༢㞳ࡋࡓࢥࣟࢽ࣮ࢆಸయೃ⿵ᰴࡋࡓࠋಸయೃ⿵ᰴࢆࡧ a924E1 ᰴ࠾ࡼࡧ KA31α ᰴࡢᇵ㣴ᾮࡑࢀࡒࢀΰྜࡋࠊ᥋ྜᏊࡀほᐹࡉࢀ࡞ࡗ ࡓࢥࣟࢽ࣮ࢆಸయᰴࡋࡓࠋసฟࡋࡓಸయᰴࡣࠊ FACS CaliburTM flow cytometer (Becton Dickinson and Co., NJ, ⡿ᅜ)ࡼࡾ DNA 㔞࠾ࡼࡧ⣽⬊ࢧࢬࢆ ☜ㄆࡋࡓࠋ ➨ 4 㡯 ྾ᶵ⬟ࡢゎᯒ᪉ἲ 㓝ẕᰴࡢ྾ᶵ⬟ࡣ 2,3,5-triphenyl-2H-tetrazolium chloride (TTC; Wako Pure Chemical Industries)ࡼࡿࢥࣟࢽ࣮ᰁⰍἲࡼࡾ Nagai(1959)ࡢሗ࿌ᚑ࠸ゎᯒ ࡋࡓࠋ ➨ 5 㡯 ࣑ࢺࢥࣥࢻࣜ DNA Ḟኻࡢゎᯒ᪉ἲ ࣑ࢺࢥࣥࢻࣜ DNA ࡢḞኻࡣ PCR ἲࡼࡾゎᯒࡋࡓࠋTotal DNA ࡣࠊDr. GenTLE® (from Yeast) High Recovery (ࢱ࢝ࣛࣂ࢜)ࢆ⏝࠸ࡓࢨ࣮ࣔࣜࢮฎ⌮ ࡼࡾᢳฟࡋࡓࠋᢳฟࡋࡓ DNA ࡣ TE ࣂࢵࣇ࣮50 μL ⁐ゎࡋࠊ㸫20°C ࡛ಖ ᏑࡋࡓࠋDNA ⃰ᗘࡣ 260 nm ࡢ྾ගᗘ࡛ ᐃࡋࡓࠋ࣑ࢺࢥࣥࢻࣜ DNA ࡢቑᖜ ࡢࡓࡵࠊSaccharomyces Genome Database (SGD, http://www.yeastgenome.org/) ࠾ 38 ࡼ ࡧ Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi/) ࢆ⏝࠸࡚ࠊ15S rRNAࠊ21S rRNAࠊCOX1ࠊCOX3ࠊCOB 㑇ఏᏊࢆᑐ㇟ࡋࡓࣉࣛ ࣐࣮ࢆタィࡋࡓ(⾲ 2)ࠋࣁ࢘ࢫ࣮࢟ࣆࣥࢢ㑇ఏᏊࡋ࡚ࡣ CDC48 㑇ఏᏊࢆ⏝ ࠸ࡓࠋ ➨ 6 㡯 㧗ᅽฎ⌮᪉ἲ 㧗ᅽฎ⌮ࡣࠊShigematsu ࡽ(2010a)ࡢሗ࿌ᚑ࠸ࠊ200 MPaࠊ20°Cࠊ0-360 s ࡢ᮲ ௳࡛⾜ࡗࡓࠋ 39 ➨ 3 ⠇ ⤖ᯝ ➨ 1 㡯 ྾ᶵ⬟ࡢゎᯒ DNA ࣐ࢡࣟࣞࡢ⥙⨶ⓗ㑇ఏᏊⓎ⌧ゎᯒࡢ⤖ᯝࡽ a924E1 ᰴ࠾࠸࡚ࠊ ࣑ࢺࢥࣥࢻࣜࡢᶵ⬟ࡀᅽຊឤཷᛶ⬟ᙳ㡪ࢆ࠼࡚࠸ࡿࡇࡀ♧၀ࡉࢀࡓࠋ ࡑࡇ࡛ TTC ᰁⰍἲࡼࡾ a924E1 ᰴࡢ࣑ࢺࢥࣥࢻࣜࡢ྾ᶵ⬟ࢆホ౯ࡋࡓࠋ ↓Ⰽࡢࢸࢺࣛࢰ࣒ࣜ࢘ሷࡣ㟁Ꮚཷᐜయࡋ࡚ാࡁࠊ㓟ⓗࣜࣥ㓟ࡀ⾜ࢃࢀࡿ ࣑ࢺࢥࣥࢻࣜ⭷㛫⭍࡛㉥Ⰽࡢ࣐࣍ࣝࢨࣥⰍ⣲㑏ඖࡉࢀࡿࠋKA31a ᰴࡢࢥࣟ ࢽ࣮ࡣ TTC ᐮኳᇵᆅࢆ㔜ᒙᚋ 20 min ࡛ࣆࣥࢡⰍᰁࡲࡾࡣࡌࡵࠊ100 min ᚋ ࡣ⃰㉥ⰍᰁⰍࡉࢀࡓ(ᅗ 1)ࠋᑐ↷ⓗࠊa924E1 ᰴࡢࢥࣟࢽ࣮ࡣ 100 min ࡀ⤒㐣 ࡋ࡚ࡶࢃࡎࣆࣥࢡⰍⰍࡃࡔࡅ࡛࠶ࡗࡓ(ᅗ 1)ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊa924E1 ᰴࡢዲẼ྾⬟ࡢάᛶࡀపୗࡋ࡚࠸ࡿࡇࢆ♧၀ࡋ࡚࠸ࡿࠋ ➨ 2 㡯 ࣑ࢺࢥࣥࢻࣜ DNA ࡢḞኻ 㓝ẕ࠾ࡅࡿ྾ᶵ⬟ࡢάᛶపୗࡣࠊ࣑ࢺࢥࣥࢻࣜ DNA ࡢḞኻࡀཎᅉ࡛࠶ ࡿࡇࡀከ࠸ࠋa924E1 ᰴࡢ྾ᶵ⬟ࡢάᛶపୗࢆᘬࡁ㉳ࡇࡋࡓ㑇ఏⓗせᅉࢆྠ ᐃࡍࡿࡓࡵࠊ࣑ࢺࢥࣥࢻࣜ DNA ࢆᑐ㇟ࡋ࡚ PCR ἲࡼࡾゎᯒࡋࡓࠋ࣑ ࢺࢥࣥࢻࣜ DNA ࡢせ࡞㑇ఏᏊ࡛࠶ࡿ 15S rRNAࠊ21S rRNAࠊCOX1ࠊCOX3ࠊ 40 COB 㑇ఏᏊࢆࢱ࣮ࢤࢵࢺࡋ࡚ࣉ࣐࣮ࣛࢆタィࡋࡓ(⾲ 2)ࠋCDC48 㑇ఏᏊࡣ ෆ㒊ᶆ‽ࡋࡓࠋࡑࡢ⤖ᯝࠊa924E1 ᰴ࠾࠸࡚ 15S rRNAࠊ21S rRNAࠊCOX3ࠊ COB 㑇ఏᏊࡢࣂࣥࢻࡣ᳨ฟࡉࢀࡓࡀࠊCOX1 㑇ఏᏊࢆྵࡴ㡿ᇦࢆࢱ࣮ࢤࢵࢺ ࡋࡓࣂࣥࢻࡣ᳨ฟࡉࢀ࡞ࡗࡓ(ᅗ 5)ࠋ ➨ 3 㡯 ಸయᰴࡢసฟ COX1 㑇ఏᏊࡢḞኻᅽຊឤཷᛶ⬟ࡢ㛵㐃ᛶࢆゎᯒࡍࡿࡓࡵࠊa ᆺ୍ಸయᰴ a924E1 ᰴ࠾ࡼࡧ α ᆺ୍ಸయ㔝⏕ᆺᰴ KA31α ᰴࡢ᥋ྜࡼࡾ 5 ᰴࡢಸయᰴࢆ సฟࡋࡓࠋసฟࡋࡓಸయᰴࡢ DNA 㔞࠾ࡼࡧ⣽⬊ࢧࢬࡣࣇ࣮ࣟࢧࢺ࣓ࢺࣜ ࣮࡛☜ㄆࡋࡓ(ᅗ 2)ࠋಸయᰴࡢ DNA 㔞(G1 ᮇ㸸2CࠊG2 ᮇ㸸4C)ࡣࠊ୍ಸయᰴ (G1 ᮇ㸸1CࠊG2 ᮇ㸸2C)ࡢ࠾ࡼࡑ 2 ಸ㔞࡛࠶ࡗࡓࠋಸయᰴࡢ G2 ᮇ(DNA 㔞㸸 4C)ࡢ⣽⬊ࡢࢧࢬࡣࠊG1 ᮇ(2C)ࡢ⣽⬊ࡢ 2 ಸ௨ୖ࡛࠶ࡾࠊྠᵝ୍ಸయᰴ ࠾࠸࡚ࡶࠊG2 ᮇ(DNA 㔞㸸2C)ࡢ⣽⬊ࡢࢧࢬࡣࠊG1 ᮇ(1C)ࡢ⣽⬊ࡢ 2 ಸ௨ୖ ࡛࠶ࡗࡓࠋಸయᰴࡢቑṪ᭤⥺ࡣᅗ 3 ♧ࡍࠋ 41 ➨ 4 㡯 ᅽຊάᛶᣲືࡢゎᯒ సฟࡋࡓಸయᰴ࠾ࡼࡧ a924E1 ᰴࠊKA31a ᰴࢆ 200 MPaࠊ20°Cࠊ0-360 s ࡢ᮲ ௳࡛㧗ᅽฎ⌮ࡋࡓࠋYPD ᐮኳᇵᆅࡼࡿࢥࣟࢽ࣮࢝࢘ࣥࢺἲࡼࡾ⏕⳦ᩘࢆ⟬ ฟࡋࡓࠋ120 sࠊ240 sࠊ360 s ࡢ᮲௳࡛㧗ᅽฎ⌮ࡋࡓࢧࣥࣉࣝࡢ⏕⳦ᩘࢆ 0 s ฎ⌮ ࢧࣥࣉࣝࡢ⏕⳦ᩘ࡛㝖ࡋ࡚⏕Ꮡ⋡ࡋࡓࠋྛฎ⌮᮲௳ࡢ⏕Ꮡ⋡ࡢ⮬↛ᑐᩘ್ࢆ ⦪㍈㧗ᅽฎ⌮㛫ࢆᶓ㍈ࣉࣟࢵࢺࡋ࡚ᅽຊάᛶ᭤⥺ࢆసᡂࡋࠊᅽຊ άᛶ㏿ᗘᐃᩘࢆ⟬ฟࡋࡓ(ᅗ 4)ࠋࡑࡢ⤖ᯝࠊKA31a ᰴẚ㍑ࡋ࡚ a924E1 ᰴࡢ ᅽຊឤཷᛶࡀ☜ㄆ࡛ࡁࡓࠋಸయᰴࡣࠊKA31a ᰴྠ⛬ᗘࡢᅽຊ⪏ᛶࢆ♧ࡍ⩌ a924E1 ᰴ༉ᩛࡍࡿᅽຊឤཷᛶࢆ♧ࡍ⩌ศࢀࡓࠋ ➨ 5 㡯 㔝⏕ᆺ࣑ࢺࢥࣥࢻࣜࡼࡿḞኻ㑇ఏᏊࡢ⿵ ྾Ḟᦆ㓝ẕᰴ㔝⏕ᆺ㓝ẕᰴࡢ᥋ྜ࠾࠸࡚ࠊ྾Ḟᦆᆺ࣑ࢺࢥࣥࢻࣜ ࡣ㔝⏕ᆺ࣑ࢺࢥࣥࢻ୍ࣜᐃࡢྜ࡛⨨ࡉࢀࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡑ ࡇ࡛ࠊa924E1 ᰴࡢ྾Ḟᦆ࠾ࡼࡧᅽຊឤཷᛶࡢ⾲⌧ᆺࡀసฟࡋࡓಸయᰴ ࡢࡼ࠺ཷࡅ⥅ࡀࢀࡿࡢホ౯ࡋࡓࠋTTC ᰁⰍࡢ⤖ᯝࠊಸయᰴࡣ TTC ࣏ࢪࢸ ࣈ(྾ᶵ⬟᭷)࠾ࡼࡧ TTC ࢿ࢞ࢸࣈ(྾ᶵ⬟పୗ) ࡢ 2 ࡘࡢ⾲⌧ᙧ㉁ࢆ♧ ࡍ⩌ศ㞳ࡋࡓ(ᅗ 1)ࠋTTC ࣏ࢪࢸࣈ⩌ࡣ KA31a ᰴྠ⛬ᗘࡢᅽຊ⪏ᛶࢆ♧ ࡍ⩌ྠࡌ࡛࠶ࡗࡓࠋྠᵝ TTC ࢿ࢞ࢸࣈ⩌ࡣ a924E1 ᰴ༉ᩛࡍࡿᅽຊឤ 42 ཷᛶࢆ♧ࡋࡓ⩌ྠࡌ࡛࠶ࡗࡓࠋࡲࡓࠊTTC ࣏ࢪࢸࣈ⩌᳨࡛ฟࡉࢀࡿ COX1 㑇ఏᏊࡀࠊTTC ࢿ࢞ࢸࣈ⩌࡛ࡣḞኻࡋ࡚࠸ࡿࡇࢆ PCR ࡼࡾ☜ㄆࡋࡓ(ᅗ 5)ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊᅽຊឤཷᛶ⬟ࡀ࣓ࣥࢹࣝᘧ㑇ఏࡍࡿࡢ࡛ࡣ࡞ࡃࠊ྾Ḟ ᦆ࣑ࢺࢥࣥࢻࣜඹ⣽⬊㉁㑇ఏࡋࡓࡇࢆ♧၀ࡍࡿࠋ 43 ➨ 4 ⠇ ⪃ᐹ ᮏ❶࡛ࡣࠊDNA ࣐ࢡࣟࣞゎᯒ࡛♧၀ࡉࢀࡓ࣑ࢺࢥࣥࢻࣜᅽຊឤཷ ᛶࡢ㛵㐃ᛶࡘ࠸࡚↔Ⅼࢆᙜ࡚࡚ゎᯒࡋࡓࠋTTC ᰁⰍࡼࡾ࣑ࢺࢥࣥࢻࣜࡢ ྾ᶵ⬟ࢆホ౯ࡋࡓࡇࢁࠊa924E1 ᰴࡢ྾ᶵ⬟ࡀపୗࡋ࡚࠸ࡿࡇࡀ♧ࡉࢀ ࡓ(ᅗ 1)ࠋ࣑ࢺࢥࣥࢻࣜ DNA ࢆᑐ㇟ࡋࡓ PCR ࡼࡗ࡚ COX1 㑇ఏᏊࢆࢥ ࣮ࢻࡍࡿ㡿ᇦࡢḞኻࡀ♧ࡉࢀࡓ(ᅗ 5)ࠋ COX1 㑇ఏᏊ௨እࡢ࣑ࢺࢥࣥࢻࣜ DNA ࡢ㑇ఏᏊ࠾࠸࡚ࡣࠊḞኻࡣ☜ㄆࡉࢀ࡞ࡗࡓ(ᅗ 5)ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊa924E1 ᰴࡢ࣑ࢺࢥࣥࢻࣜ྾ᶵ⬟ࡢపୗࡀ COX1 㑇ఏᏊࡢḞኻ㉳ᅉࡋ࡚࠸ࡿࡇ ࢆ♧၀ࡋ࡚࠸ࡿࠋ୍⯡ⓗ྾Ḟᦆ㓝ẕᰴࡣ࡚ࡢ࣑ࢺࢥࣥࢻࣜ DNA ࢆḞᦆ ࡋ࡚࠸ࡿࡀࠊa924E1 ᰴࡣ COX1 㑇ఏᏊࡢࡳࢆḞኻࡋࡓ㠀ᖖ⛥࡞྾Ḟᦆᰴ ࡛࠶ࡾࠊ࣑ࢺࢥࣥࢻࣜ྾ᶵ⬟㛵ࡍࡿ◊✲࠾࠸࡚ࡶ᭷┈࡞ኚ␗ᰴ࡛࠶ ࡿࠋ సฟࡋࡓಸయᰴ࠾࠸࡚ࠊCOX1 㑇ఏᏊࡢḞኻࡣࠊ࣑ࢺࢥࣥࢻࣜඹ ⣽⬊㉁㑇ఏࡋࡓ(ᅗ 1)ࠋCOX1 㑇ఏᏊࢆḞኻࡋࡓ࣑ࢺࢥࣥࢻࣜࢆᣢࡘ྾ᶵ⬟ ࡀపୗࡋࡓಸయᰴࡣࠊCOX1 㑇ఏᏊḞኻ࣑ࢺࢥࣥࢻࣜࡀ㔝⏕ᆺ࣑ࢺࢥࣥࢻ ࣜ⨨ࡁ࠼ࡽࢀࡓ྾ᶵ⬟ࢆ⥔ᣢࡋࡓಸయᰴẚ㍑ࡋ࡚ࠊ᭷ព㧗࠸ᅽ ຊឤཷᛶࢆ♧ࡋࠊࡑࡢᅽຊឤཷᛶ⬟ࡣ a924E1 ᰴ༉ᩛࡍࡿࡇࡀ♧ࡉࢀࡓ(ᅗ 44 4)ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊCOX1 㑇ఏᏊࡢḞኻࡀ a924E1 ᰴᅽຊឤཷᛶ⬟ࢆࡋ ࡚࠸ࡿ୍ᅉ࡛࠶ࡿࡇࢆ♧ࡋ࡚࠸ࡿࠋ࣑ࢺࢥࣥࢻࣜᶵ⬟ࡀᅽຊឤཷᛶ⬟ᙉ ࡃ㛵㐃ࡍࡿ࠸࠺ࡇࢀࡽࡢ⤖ᯝࡣ๓ࡀ↓࠸ࡀࠊᇶ♏ⓗ࡞ᅽຊឤཷᛶᶵᵓࢆ⌮ ゎࡍࡿࡓࡵࡢࣈࣞࢡࢫ࣮ࣝ࡞ࡿྍ⬟ᛶࡀ࠶ࡿࠋ ࣑ࢺࢥࣥࢻࣜࡣࠊATP ࡸ NADHࠊᵝࠎ࡞࣑ࣀ㓟ࡢ୰㛫⏘≀➼ࢆ⏕ᡂࡍࡿ せ࡞௦ㅰ⤒㊰ࡢ୰ᚰⓗ࡞ᙺࢆᢸ࠺ࠋ࠸ࡃࡘࡢྜ≀ࡣቑṪᚲ㡲࡛ࡣ࡞ ࠸ࡀࠊ࣑ࢺࢥࣥࢻࣜᶵ⬟ࡢపୗࡼࡗ࡚せ࡞௦ㅰࡀṆࡍࡿࡇࡣࠊከࡃࡢ ࢫࢺࣞࢫ⪏ᛶࢆ㜼ᐖࡍࡿࡇࡘ࡞ࡀࡿࠋ࠼ࡤࠊࣉࣟࣜࣥࠊࣝࢠࢽࣥࠊࢢࣝ ࢱ࣑ࣥ㓟ࠊᒀ⣲➼ࡢ௦ㅰ㛵㐃ࡍࡿ㓝⣲ࡣ࣑ࢺࢥࣥࢻࣜᒁᅾࡋ࡚࠾ࡾࠊࡇࢀ ࡽࡣ㓟ࢫࢺࣞࢫࠊ㧗 ࢫࢺࣞࢫࠊ⤖⼥ゎࢫࢺࣞࢫ➼ࡢࢫࢺࣞࢫ⪏ᛶᐤࡋ ࡚࠸ࡿࠋࡇࢀࡽࡢ⤖ᯝࡣࠊa924E1 ᰴᅽຊឤཷᛶ⬟ࢆࡋࡓせᅉࡋ࡚ࠊ COX1 㑇ఏᏊࡢḞኻࡼࡿ࣑ࢺࢥࣥࢻࣜࡢᶵ⬟㉳ᅉࡍࡿ┤᥋ⓗ࡞ᙳ㡪 ࡛ࡣ࡞ࡃࠊ࣑ࢺࢥࣥࢻࣜࡢᶵ⬟క࠺ࢫࢺࣞࢫ⪏ᛶᐤࡍࡿ௦ㅰ⏘≀ ࡢῶᑡࡼࡗ࡚ᵝࠎ࡞ࢫࢺࣞࢫᑐࡍࡿ⪏ᛶࡀపୗࡋࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ ୍⯡ⓗ྾Ḟᦆ㓝ẕᰴࡣ࡚ࡢ࣑ࢺࢥࣥࢻࣜ DNA ࢆḞᦆࡋ࡚࠾ࡾࠊࡑࡢ ࢚ࢱࣀ࣮ࣝⓎ㓝⬟࠾ࡼࡧቑṪ⬟ࡣ㔝⏕ᆺᰴẚ㍑ࡋ࡚ࡁࡃῶᑡࡍࡿࠋࡋࡋ ࡞ࡀࡽࠊᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴࡣ COX1 㑇ఏᏊḞኻ㉳ᅉࡍࡿ࣑ࢺࢥࣥ ࢻࣜᶵ⬟ࢆᘬࡁ㉳ࡇࡋ࡚࠸ࡿࡶ㛵ࢃࡽࡎࠊKA31a ᰴྠ➼ࡢ࢚ࢱࣀ࣮ 45 ࣝⓎ㓝⬟(Shigematsu et al., 2010a)࠾ࡼࡧቑṪ⬟(ᅗ 3)ࢆ᭷ࡍࡿࠋBrauer ࡽ (2005)ࡼࡗ࡚ ࠊࢢ ࣝࢥ࣮ ࢫ⃰ ᗘࡀ 0.5%௨ ୖࡢ ᮲௳ ࡛ᇵ㣴 ࡍࡿሙ ྜࠊ S. cerevisiae ࡢቑṪࡣ࢚ࢱࣀ࣮ࣝⓎ㓝౫Ꮡࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿࡇ ࡽࠊa924E1 ᰴ࠾ࡼࡧ KA31a ᰴࡣ࢚ࢱࣀ࣮ࣝⓎ㓝ࡍࡿࡇࡼࡾぢࡅୖྠ ➼ࡢቑṪ⬟ࢆ᭷ࡋ࡚࠸ࡓ⪃࠼ࡽࢀࡿࠋࡲࡓࠊࡇࢀࡽࡢ⤖ᯝࡣࠊCOX1 㑇ఏᏊࡢ Ḟኻ㉳ᅉࡍࡿ௦ㅰࡢኚࡀ࢚ࢱࣀ࣮ࣝⓎ㓝⬟ᙳ㡪ࢆཬࡰࡉࡎᅽຊឤཷᛶ ⬟ࢆࡋ࡚࠸ࡿࡇࢆ♧၀ࡋ࡚࠸ࡿࡇࡣࠊPReF ᢏ⾡ࡢᐇ⏝ྥࡅ࡚᭷┈ ࡛࠶ࡿࠋ ᮏ❶ࡢ⤖ᯝࢆࡲࡵࡿࠊa924E1 ᰴࡢᅽຊឤཷᛶ⬟ࡣ࣑ࢺࢥࣥࢻࣜࡢᶵ⬟ ࡼࡾࡉࢀࡓ⪃࠼ࡽࢀࡿࠋࡋࡋ࡞ࡀࡽࠊᅽຊឤཷᛶࢆࡍࡿᶵᵓ ࡢ┤᥋ⓗ࡞ドᣐࡣ᫂ࡽ࡞ࡽ࡞ࡗࡓࡀࠊa924E1 ᰴࡢ࣑ࢺࢥࣥࢻࣜࢆࡢ 㓝ẕᰴࡢ࣑ࢺࢥࣥࢻࣜ⿵ࡍࡿࡇ࡛ᐜ᫆⏘ᴗ⏝㓝ẕᰴᅽຊឤཷᛶ⬟ ࢆࡍࡿࡇࡀᮇᚅ࡛ࡁࡿࠋ 46 ➨ 4 ❶ ࣓ࢱ࣑࣎ࣟࢡࢫࡼࡿᅽຊឤཷᛶᶵᵓࡢゎᯒ ➨ 1 ⠇ ⥴ゝ ➨ 1 㡯 ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒ ࣓ࢱ࣑࣎ࣟࢡࢫࡣࠊ᭷ᶵྜ≀ࠊ࣑ࣀ㓟ࠊ᰾㓟➼ࡢ⣽⬊ෆ௦ㅰ⏘≀ࢆࢱ࣮ࢤ ࢵࢺࡋࡓ⥙⨶ⓗゎᯒᢏ⾡࡛࠶ࡿ(⚟ᓮ, 2013)ࠋ࣓ࢱ࣑࣎ࣟࢡࢫࡢࡁ࡞≉ᛶࡣ ࡑࡢ୍⯡ᛶ࡛࠶ࡿࠋDNA ࡸ RNA ࢆ⥙⨶ⓗゎᯒࡍࡿࢤࣀ࣑ࢡࢫࠊࢱࣥࣃࢡ㉁ ࢆ⥙⨶ⓗゎᯒࡍࡿࣉࣟࢸ࣑࢜ࢡࢫ➼ࡼࡿゎᯒ࡛ࡣࠊࡑࡢゎᯒࢧࣥࣉࣝ≉ ᭷ࡢሷᇶ㓄ิ➼ࡢሗࡀᚲせ࡛࠶ࡿࠋࡑࡢ୍᪉ࠊ࣓ࢱ࣑࣎ࣟࢡࢫࡢゎᯒᑐ㇟ࡣ௦ ㅰ⏘≀࡛࠶ࡾࠊᙜ↛ࡢࡇ࡞ࡀࡽࡑࢀࡽࡣࣄࢺ࡛ࡶ⭠⳦࡛ࡶ⏕≀㛫࡛ᛶ ࢆ᭷ࡍࡿྜ≀࡛࠶ࡿሙྜࡀከ࠸ࠋࡋࡋࠊ࣓ࢱ࣑࣎ࣟࢡࢫ࡛ゎᯒ࡛ࡁࡿ௦ㅰ⏘ ≀ᶵ⬟ᛶࡀ࠶ࡿࡣ㝈ࡽ࡞࠸ࡓࡵࠊ⏕≀Ꮫⓗ࡞⌮⏤ࡅࡀ㞴ࡋ࠸࠸࠺ၥ 㢟ࡶ࠶ࡿࠋ࠼ࡤࠊ࠶ࡿ௦ㅰ⏘≀ࡀቑຍࡋࡓሙྜࠊᚲせ࡞ࡗࡓࡓࡵ✚ࡋࡓ ࡢࠊせ࡞ࡗࡓࡢࡽ✚ࡋࡓࡢࡢゎ㔘ࡣࠊูࡢᐇ㦂⤖ᯝ࡛⌮⏤ࡅࢆ⾜ ࠺ᚲせࡀ࠶ࡿ(㔝ᮧ, ᒾᶫ, 2013)ࠋ 47 ➨ 2 㡯 ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒࡢᛂ⏝ ከ✀ከᵝ࡞ྜ≀ࢆศᯒࡍࡿ࣓ࢱ࣑࣎ࣟࢡࢫ᭱ࡶࡼࡃ⏝࠸ࡽࢀࡿᢏ⾡ࡣࠊ ㉁㔞ศᯒ࡛࠶ࡿࠋゎീᗘ⌧ᛶඃࢀࡓ࢞ࢫࢡ࣐ࣟࢺࢢࣛࣇ࣮㉁㔞ศᯒ (GC/MS)ࡸᾮయࢡ࣐ࣟࢺࢢࣛࣇ࣮㉁㔞ศᯒ(LC/MS)ࠊ⢾ࣜࣥ㓟ࡸ᰾㓟➼᭷ຠ ࡞࢟ࣕࣆ࣮ࣛࣜ㟁ẼὋື㉁㔞ศᯒ(CE/MS)➼ࡢᡭἲࡀࡼࡃ▱ࡽࢀ࡚࠸ࡿ(⚟ᓮ, 2013)ࠋ Tanaka ࡽ(2007)ࡣࠊ㓝ẕࡢ࢝ࢻ࣑࣒࢘ࢫࢺࣞࢫᛂ⟅ࢆゎᯒࡍࡿࡓࡵ࢟ࣕࣆࣛ ࣮ࣜ㟁ẼὋື㉁㔞ศᯒἲ(CE/MS)ࢆ⏝࠸ࡓ⣽⬊ෆ௦ㅰ⏘≀ࡢ⥙⨶ⓗゎᯒࢆ⾜࠸ࠊ ࢫࢺࣞࢫᛂ⟅ゎᯒ࠾ࡅࡿ࣓ࢱ࣑࣎ࣟࢡࢫࡢ᭷⏝ᛶࢆ♧ࡋࡓࠋࡲࡓࠊࢤࣀ࣑ࢡࢫ ࣓ࢱ࣑࣎ࣟࢡࢫࢆ⤌ࡳྜࢃࡏࡿࡇ࡛ࠊᶵ⬟ሗ௦ㅰሗࢆྠゎᯒࡍ ࡿࡇࡶሗ࿌ࡉࢀ࡚࠸ࡿ(⏣୰ࡽ, 2013)ࠋᶵ⬟ሗࡣཎᅉ࡛࠶ࡾࠊ௦ㅰሗࡣ⤖ ᯝ࡛࠶ࡿࠋࡇࢀࡽࢆ⤌ࡳྜࢃࡏࡿࡇ࡛ࠊࡢࡼ࠺࡞㑇ఏᏊࡢⓎ⌧ࡢኚࡀཎᅉ ࡞ࡗ࡚ࠊࢫࢺࣞࢫ➼㐺ᛂࡍࡿ⤖ᯝ࡞ࡿࡢࠊࡼࡾ῝ࡃ⪃ᐹࡍࡿࡇࡀ࡛ࡁ ࡿࠋ ➨ 3 㡯 ࣝࢠࢽࣥ ࣝࢠࢽࣥࡣࢢࢽࢪࣥ㢮ఝࡋࡓ࣑ࣀ㓟ഃ㙐ࢆ᭷ࡍࡿሷᇶᛶ࣑ࣀ㓟ࡢ 1 ✀࡛࠶ࡿ(Hamada and Shirali, 2007)ࠋࢢࢽࢪࣥࡣࢱࣥࣃࢡ㉁ࡢจ㞟ࢆᢚไ 48 ࡍࡿኚᛶ࡛࠶ࡿࡓࡵࠊࢢࢽࢪࣥ㢮ఝࡋࡓഃ㙐ࢆᣢࡘࣝࢠࢽࣥࡶจ㞟ࡋ ࡓࢱࣥࣃࢡ㉁ࡢ࣮ࣜ࣍ࣝࢹࣥࢢࢆ⿵ຓࡍࡿ᭷ᮃ࡞ῧຍ≀࡛࠶ࡿ(Tsumoto et al., 2004; Lyutoya et al., 2007)ࠋࡑࢀᨾࠊࣝࢠࢽࣥࡣ㓝ẕ࠾࠸࡚㓟ࡸ ⤖ᑐࡍࡿࢫࢺࣞࢫ⪏ᛶ㛵ࡍࡿࡇࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Morita et al., 2002; Nishimura et al., 2010)ࠋ㧗ᅽࡣ㠀ඹ᭷⤖ྜࡢᏳᐃࢆᘬࡁ㉳ࡇࡋ࡚ࢱࣥࣃࢡ ㉁ࡢኚᛶࢆಁ㐍ࡍࡿࠋࡋࡋ࡞ࡀࡽࠊࣝࢠࢽࣥࡢ㧗ᅽࢫࢺࣞࢫᑐࡍࡿຠᯝࡣ ᮍࡔ࠶ࡁࡽ࡞ࡗ࡚࠸࡞࠸ࠋ ➨ 4 㡯 ᐇ㦂┠ⓗ ➨ 2 ❶࠾ࡼࡧ➨ 3 ❶ࡢ⤖ᯝࡽࠊa924E1 ᰴࡢᅽຊឤཷᛶ⬟ࡀ COX1 㑇ఏᏊ ࡢḞኻ㉳ᅉࡍࡿ࣑ࢺࢥࣥࢻࣜࡢᶵ⬟ࡼࡾࡉࢀࡓࡇࡀ♧၀ࡉࢀ ࡓࠋᮏ㡯࡛ࡣࠊ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒࡼࡾࠊ࣑ࢺࢥࣥࢻࣜࡢᶵ⬟ࡼࡾᅽ ຊឤཷᛶࢆᘬࡁ㉳ࡇࡍ࣓࢝ࢽࢬ࣒ࢆゎᯒࡋࡓࠋࡲࡓࠊ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒࡼࡾ ᵝࠎ࡞ࢫࢺࣞࢫ⪏ᛶᐤࡍࡿࣝࢠࢽࣥࡢῶᑡࡀ♧ࡉࢀࡓࡓࡵࠊࣝࢠࢽ ࣥࡢᅽຊ⪏ᛶࡢᐤࡘ࠸࡚ࡶేࡏ࡚ゎᯒࡋࡓࠋ 49 ➨ 2 ⠇ ᐇ㦂ᮦᩱ࠾ࡼࡧᐇ㦂᪉ἲ ➨ 1 㡯 ⏝⳦ᰴ ᮏᐇ㦂ࡣࠊᐇ㦂ᐊ㓝ẕᰴ S. cerevisiae KA31a ᰴ࠾ࡼࡧᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴࡢᐃᖖᮇ⣽⬊ࢆ⏝ࡋࡓ(⾲ 1)ࠋᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴࡣࠊ⣸እ ⥺↷ᑕࡼࡿ✺↛ኚ␗ࡼࡾྲྀᚓࡋࡓ(Shigematsu et al., 2010a)ࠋࡇࢀࡽࡢ㓝ẕᰴ ࡣࠊ᪂₲⸆⛉Ꮫ 㔜ᯇ ᩍᤵࡼࡾศࡋ࡚࠸ࡓࡔ࠸ࡓࠋ ➨ 2 㡯 ᇵ㣴᮲௳ YPD ᇵᆅ(2.0% peptoneࠊ1.0% yeast extract; Becton Dickinson and Co., NJ, USAࠊ 2.0% glucose; ග⣧⸆ᕤᴗ, 㜰, ᪥ᮏ)ࢆ⏝࠸࡚ࠊ30°Cࠊ48 h ࡢ᮲௳࡛┞ᇵ 㣴ࡋࡓᐃᖖᮇ⣽⬊ࢆᐇ㦂౪ࡋࡓࠋቑṪ᭤⥺ࡣ Hasegawa ࡽ(2012)ࡢ᪉ἲᚑ࠸ࠊ Bio Microplate Reader HiTSTM (Scinics, Ⲉᇛ, ᪥ᮏ)ࢆ⏝࠸࡚ 30°Cࠊ120 h ࡢ᮲௳࡛ ᐃࡋࡓࠋ ➨ 3 㡯 ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒ᪉ἲ ௦ㅰ⏘≀ࡢᢳฟἲࡣ Human Metabolomics Technologies (HMT; ᒣᙧ, ᪥ᮏ)ࡢࣉ ࣟࢺࢥࣝᚑࡗࡓࠋCE/MS ࡼࡿ௦ㅰ⏘≀ࡢ᳨ฟࡣ HMT ጤクࡋࡓࠋa924E1 50 ᰴࡽ᳨ฟࡋࡓྛྜ≀ࡢ┦ᑐ್ࡣ KA31a ᰴࡢࡑࢀࡽẚ㍑ࡋ࡚ 2 ಸ௨ୖ㧗࠸ ್ࢆ♧ࡍྜ≀ࠊ༙ศ௨ୗࡢప࠸್ࢆ♧ࡍྜ≀ࠊ್ࡢኚࡢ↓࠸ྜ≀ศ㢮 ࡋࠊ࢙࢘ࣝࢳࡢ t ᳨ᐃࡼࡾ⤫ィⓗゎᯒࡋࡓࠋKA31a ᰴẚ㍑ࡋ࡚᭷ពኚື ࡋࡓྜ≀ࡢ௦ㅰ⤒㊰ࡣ KEGG (http://www.genome.jp/kegg/pathway.html)ᚑࡗ ࡚ゎᯒࡋࡓࠋ ➨ 4 㡯 ௦ㅰ⤒㊰ࡢゎᯒ᪉ἲ total RNA ࡣࠊFast RNA ® Pro Red Kit (MP Biomedicals, CA, USA)ࡼࡾᢳฟࡉ ࢀࠊRNeasy ® Mini Kit (Qiagen, Hilden, Germany)ࡼࡾ⢭〇ࡋࡓࠋcDNA ࡣࠊᢳฟ ࡋࡓ RNA ࡽ ReverTra Ace® qPCR RT Master Mix (Toyobo, 㜰, ᪥ᮏ)ࡼࡾ㏫ ㌿ࡋࡓࠋࣝࢠࢽࣥ⏕ྜᡂ㛵㐃ࡍࡿ 4 㑇ఏᏊᑐࡋ࡚ࣉ࣐࣮ࣛࢆタィࡋ ࡓ(⾲ 2)ࠋࡇࢀࡽࡢ㑇ఏᏊࡢ࠺ࡕ 3 㑇ఏᏊࡣ DNA ࣐ࢡࣟࣞゎᯒࡼࡾࠊ ࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺࡋࡓ㑇ఏᏊ(ARG3ࠊARG1ࠊARG4 㑇ఏᏊ)ࢆᑐ㇟ࡋࡓࠋARG8 㑇ఏᏊࡣ᭷ពᕪࡀㄆࡵࡽࢀ࡞ࡗࡓࡀࠊࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺࡋ࡚࠾ࡾࠊࣝࢠࢽ ࣥ⏕ྜᡂ㔜せ࡞㓝⣲ࢆࢥ࣮ࢻࡋ࡚࠸ࡿࡓࡵ㑅ᢥࡋࡓࠋPCR ࡣ GoTaq Green Master Mix (Promega, WI, USA)ࢆ⏝࠸࡚ࠊ๓㏙ࡢ᮲௳࡛ቑᖜࡋࡓࠋ 51 ➨ 5 㡯 㧗ᅽฎ⌮᪉ἲ 㧗ᅽࢫࢺࣞࢫ⪏ᛶࡢࣝࢠࢽࣥࡢᙳ㡪ࢆゎᯒࡍࡿࡓࡵࠊࣝࢠࢽࣥῧຍ (10-500 mM)࠶ࡿ࠸ࡣᮍῧຍࡢ᮲௳࠾࠸࡚ᇵ㣴ᚋࠊ200 MPaࠊ0-4°Cࠊ360 s ࡢ᮲ ௳࡛㧗ᅽฎ⌮ࡋࡓࠋ㧗ᅽฎ⌮ࡣࣁࣥࢻ࣏ࣥࣉᆺ㧗ᅽ⨨ HP-500 (Syn Corporation, ி㒔, ᪥ᮏ)ࢆ⏝࠸ࡓ௨እࡣࠊShigematsu ࡽ(2010a)ࡢሗ࿌ᚑࡗࡓࠋ㧗ᅽฎ⌮ᚋ ⏕⌮㣗ሷỈ࡛ 10-100 ಸᕼ㔘ࡋ࡚ࠊ5 μL ࡎࡘ YPD ᐮኳᇵᆅࢫ࣏ࢵࢺࡋࠊ 30°C ࡛㟼⨨ᇵ㣴ࡋࡓࠋ 52 ➨ 3 ⠇ ⤖ᯝ ➨ 1 㡯 ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒ CE/MS ゎᯒࡼࡾࠊ250 ✀㢮ࡢྜ≀ࢆ᳨ฟࡋࡓ(⾲ 8)ࠋᮏ◊✲࡛ࡣ a924E1 ᰴ ࠾࠸࡚ KA31a ᰴẚ㍑ࡋ࡚┦ᑐⓗῶᑡࡋࡓྜ≀╔┠ࡋࡓࠋࢢࣝࢱ࣑ࣥ 㓟ࠊࢫࣃࣛࢠࣥ㓟ࠊࢢࣝࢱ࣑ࣥࠊ࢜ࣝࢽࢳࣥࠊࣝࢠࢽࣥ➼ࡢ࣑ࣀ㓟ࢆྵࡴ 48 ✀㢮ࡢྜ≀ࡣࠊKA31a ᰴࡢࡑࢀࡽẚ㍑ࡋ࡚᭷ពప࠸್࡛࠶ࡗࡓ(p < 0.05)ࠋࢭࢳࣝ CoAࠊࢥࢽࢵࢺ㓟ࠊࣇ࣐ࣝ㓟ࠊࢯࢡ࢚ࣥ㓟➼ࡢ TCA ࢧࢡ ࣝ㛵㐃ࡍࡿྜ≀➼ࡢ 59 ✀㢮ࡢྜ≀ࡣࠊa924E1 ᰴ࡛ࡣ᳨ฟࡉࢀ࡞ࡗࡓࠋ ࡲࡓࠊα-ࢣࢺࢢࣝࢱࣝ㓟➼ࡢ࠸ࡃࡘࡢྜ≀ࡣ a924E1 ᰴ࠾ࡼࡧ KA31a ᰴࡢ୧ ᰴ᳨࡛ฟࡉࢀ࡞ࡗࡓࠋࡲࡓ a924E1 ᰴ࠾࠸࡚ࠊ᳨ฟࡉࢀࡓ TCA ࢧࢡࣝ 㛵㐃ࡍࡿྜ≀ࡢ࠺ࡕࠊࢥࣁࢡ㓟ࡢࡳࡀእⓗቑຍࡋ࡚࠸ࡓࠋࡇࢀࡽࡢ a924E1 ᰴ࠾࠸࡚ኚືࡋࡓྜ≀ࡣࠊࣝࢠࢽࣥ⏕ྜᡂ㛵㐃ࡋ࡚࠸ࡿ(⾲ 8)ࠋ ➨ 2 㡯 ࣝࢠࢽࣥ௦ㅰゎᯒ⤒㊰㛵ࡍࡿ㑇ఏᏊࡢゎᯒ ⾲ 9 ♧ࡉࢀࡓࢢࣝࢱ࣑ࣥࠊࢫࣃࣛࢠࣥ㓟ࠊࢢࣝࢱ࣑ࣥ㓟ࠊ࢜ࣝࢽࢳࣥࡣࠊ ࣝࢠࢽࣥࡢ⏕ྜᡂ㛵㐃ࡍࡿ࣑ࣀ㓟࡛࠶ࡿࠋࣝࢠࢽࣥࡣࢫࢺࣞࢫᛂ⟅ 㔜せ࡞ᙺࢆᢸ࠺࣑ࣀ㓟࡛࠶ࡿࡓࡵࠊࡑࡢ⏕ྜᡂ㛵㐃ࡍࡿ㓝⣲⩌ࢆࢥ࣮ࢻ 53 ࡍࡿ㑇ఏᏊࡢⓎ⌧ࢆホ౯ࡍࡿࡇࡣ㔜せ࡛࠶ࡿࠋᮏ㡯࡛ࡣࠊࣝࢠࢽࣥ⏕ྜᡂ㓝 ⣲ࢆࢥ࣮ࢻࡍࡿ ARG8ࠊARG3ࠊARG1ࠊARG4 㑇ఏᏊࡢⓎ⌧ࢆ RT-PCR ἲࡼࡾ ゎᯒࡋࡓࠋෆ㒊ᶆ‽ࡋ࡚ࢡࢳࣥࢆࢥ࣮ࢻࡍࡿ ACT1 㑇ఏᏊࢆ⏝࠸ࡓࠋࡑࡢ⤖ ᯝࠊゎᯒࡋࡓ 4 㑇ఏᏊ࠾࠸࡚ࠊKA31a ᰴ࡛ࡣⓎ⌧ࡀ☜ㄆ࡛ࡁࡓࡀࠊa924E1 ᰴ ࡛ࡣⓎ⌧ࢆ☜ㄆ࡛ࡁ࡞ࡗࡓ(ᅗ 6)ࠋ ➨ 3 㡯 ࣝࢠࢽࣥࡢᅽຊάᛶࡢᐤ ࣝࢠࢽࣥࡢ㧗ᅽࢫࢺࣞࢫ⪏ᛶࡢᙳ㡪ࢆゎᯒࡍࡿࡓࡵࠊࢫ࣏ࢵࢺヨ㦂 ࡼࡾࣝࢠࢽࣥࡢ᭷↓ࡼࡿ a924E1 ᰴࡢᅽຊឤཷᛶ⬟ࢆホ౯ࡋࡓࠋࣝࢠࢽࣥ ↓ῧຍ᮲௳࠾࠸࡚ࠊKA31a ᰴ࡛ࡣ㧗ᅽฎ⌮ᚋ⣽⬊ࡢቑṪࡀほᐹࡉࢀࡓࠋ୍ ᪉ࠊa924E1 ᰴ࡛ࡣቑṪࡀほᐹ࡛ࡁ࡞ࡗࡓࠋa924E1 ᰴ࠾࠸࡚ࠊᇵ㣴ࣝ ࢠࢽࣥࢆῧຍࡋ࡚ᇵ㣴ࡋࡓሙྜࠊ10 mM ௨ୖࡢ᮲௳࠾࠸࡚ࠊ㧗ᅽฎ⌮ᚋ⣽ ⬊ࡢቑṪࢆほᐹࡋࡓࠋKA31a ᰴ࠾࠸࡚ࡣࠊ࡚ࡢ᮲௳࡛⣽⬊ࡢቑṪࡀほᐹࡉ ࢀࡓ(ᅗ 8)ࠋ 54 ➨ 4 ⠇ ⪃ᐹ ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒࡢ⤖ᯝࠊ⾲ 8 ♧ࡋࡓࡼ࠺ a924E1 ᰴ࠾࠸࡚ TCA ࢧ ࢡࣝࡸࣝࢠࢽࣥ⏕ྜᡂ㛵㐃ࡍࡿྜ≀ࡀ┦ᑐⓗῶᑡࡋ࡚࠸ࡓ(⾲ 9)ࠋ TCA ࢧࢡࣝ㛵㐃ࡍࡿྜ≀ࡢ࠺ࡕࠊࢭࢳࣝ CoAࠊࢥࢽࢵࢺ㓟ࠊࣇ࣐ࣝ 㓟ࠊࢯࢡ࢚ࣥ㓟➼ࡢྜ≀ࡣῶᑡࡋ࡚࠸ࡓࡀࠊࢥࣁࢡ㓟ࡔࡅࡀእⓗቑຍࡋ ࡓ(⾲ 8)ࠋࢥࣁࢡ㓟ࡣࠊ࣑ࢺࢥࣥࢻࣜ⭷㛫⭍ᒁᅾࡍࡿ㟁Ꮚఏ㐩㙐ࡢ」ྜయ II ࡢゐ፹స⏝ࡼࡗ࡚ࣇ࣐ࣝ㓟㓟ࡉࢀࡿࠋa924E1 ᰴ࠾ࡅࡿࢥࣁࢡ㓟ࡢቑຍ ࠾ࡼࡧࣇ࣐ࣝ㓟ࡢῶᑡࡣ࣑ࢺࢥࣥࢻࣜ㟁Ꮚఏ㐩㙐ࡢᶵ⬟ࢆ♧၀ࡋ࡚࠸ࡿࠋ DNA ࣐ࢡࣟࣞゎᯒࡢ⤖ᯝࡽ a924E1 ᰴ࠾࠸࡚ࠊTCA ࢧࢡࣝ㛵㐃 ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ 31 㑇ఏᏊࡢ࠺ࡕ 8 㑇ఏᏊ(CIT2ࠊCIT3ࠊGDH1ࠊ GDH3ࠊIDP2ࠊSDH1ࠊSDH2ࠊSHH3 㑇ఏᏊ)ࡢⓎ⌧ࡀ᭷ពࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺ ࡋ࡚࠸ࡓࠋ≉㟁Ꮚఏ㐩㙐ࡢ」ྜయ II (ࢥࣁࢡ㓟ࢆࣇ࣐ࣝ㓟௦ㅰࡍࡿ㓝⣲)㛵 㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢ༙ᩘࡀࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺࡋ࡚࠸ࡓ (SDH1ࠊSDH2ࠊSHH3 㑇ఏᏊ)ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊCOX1 㑇ఏᏊࡢḞኻ㉳ᅉࡍࡿ ࣑ࢺࢥࣥࢻࣜࡢᶵ⬟࠾ࡼࡧࡑࢀࡼࡿ࢚ࢿࣝࢠ࣮㊊ࡼࡗ࡚ࠊTCA ࢧ ࢡࣝࡀṆࡋ࡚࠸ࡿࡇࢆ♧၀ࡋ࡚࠸ࡿࠋᐇ㝿ࠊTTC ᰁⰍἲࡼࡾ྾ᶵ ⬟ࢆホ౯ࡋࡓ⤖ᯝࡶࡑࢀࡽࡢ⤖ᯝࢆᨭᣢࡋࡓ(ᅗ 1)ࠋ ⾲ 8 ♧ࡍࡼ࠺ࠊa924E1 ᰴ࠾࠸࡚ࣝࢠࢽࣥࡢࡳ࡛ࡣ࡞ࡃࢢࣝࢱ࣑ࣥ㓟 55 ࡸࢢࣝࢱ࣑ࣥࡶ᭷ពῶᑡࡋ࡚࠸ࡿࠋࢢࣝࢱ࣑ࣥ㓟ࡢ⏕ྜᡂ㛵ࡍࡿ㓝⣲ࢆࢥ ࣮ࢻࡍࡿ㑇ఏᏊ(GDH1ࠊGDH3 㑇ఏᏊ)ࡶࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺࡋ࡚࠸ࡓࡇࡽࠊ TCA ࢧࢡࣝࡢṆࡣࠊࣝࢠࢽࣥ⏕ྜᡂࡢ୰㛫≀㉁࡛࠶ࡿࢢࣝࢱ࣑ࣥ㓟ࡢᯤ Ῥࢆᘬࡁ㉳ࡇࡋ࡚࠸ࡿྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋࣝࢠࢽࣥࡢ⏕ྜᡂࡣࢢࣝࢱ࣑ ࣥ㓟࢚ࢿࣝࢠ࣮ࡀᚲ㡲࡛࠶ࡿࡓࡵࠊa924E1 ᰴ࠾࠸࡚ࣝࢠࢽࣥ⏕ྜᡂ⤒ ㊰ࡢάᛶࡣᚲ↛ⓗపୗࡍࡿࡔࢁ࠺ࠋDNA ࣐ࢡࣟࣞゎᯒࡢ⤖ᯝࠊࣝࢠ ࢽࣥࡢ⏕ྜᡂ㛵㐃ࡍࡿ㓝⣲ࢆࢥ࣮ࢻࡍࡿ 21 㑇ఏᏊࡢ࠺ࡕ 8 㑇ఏᏊࡀࢲ࢘ࣥࣞ ࢠ࣮ࣗࣞࢺࡋ࡚࠸ࡓ(ARG1ࠊARG3ࠊARG4ࠊARG7ࠊCPA1ࠊMAK31ࠊPUT1ࠊVBA1 㑇ఏᏊ)ࠋDNA ࣐ࢡࣟࣞゎᯒࡢ⤖ᯝࢆ☜ㄆࡋࠊࣝࢠࢽࣥࡢ┦ᑐⓗ࡞ῶᑡ ࡢཎᅉࢆホ౯ࡍࡿࡓࡵࠊࢢࣝࢱ࣑ࣥ㓟ࡽࣝࢠࢽࣥࢆ⏕ྜᡂࡍࡿせ࡞㓝 ⣲⩌ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢⓎ⌧ࢆ RT-PCR ࡼࡾゎᯒࡋࡓࠋࡑࡢ⤖ᯝࠊa924E1 ᰴ ࠾࠸࡚ࣝࢠࢽࣥ⏕ྜᡂ㛵㐃ࡍࡿ㑇ఏᏊࡢⓎ⌧ࡢῶᑡࡀ☜ㄆࡉࢀࡓ(ᅗ 6)ࠋ ࡇࢀࡽࡢ⤖ᯝࡣࠊTCA ࢧࢡࣝࡢṆࡼࡾࠊࢢࣝࢱ࣑ࣥ㓟ࡽࣝࢠࢽࣥࢆ ⏕ྜᡂࡍࡿ௦ㅰ⤒㊰ࡢάᛶࡢపୗࡋ࡚࠸ࡿࡇࢆ♧၀ࡋ࡚࠸ࡿ(ᅗ 7)ࠋ ࣝࢠࢽࣥࡀ㧗ᅽ⪏ᛶཬࡰࡍᙳ㡪ࢆホ౯ࡍࡿࡓࡵࠊࣝࢠࢽࣥῧຍࡲࡓ ࡣᮍῧຍࡢ᮲௳࡛ᇵ㣴ࡋࡓᚋ㧗ᅽฎ⌮ࢆࡋࠊࢫ࣏ࢵࢺヨ㦂ࡼࡾቑṪࢆ☜ ㄆࡋࡓࠋࣝࢠࢽࣥᮍῧຍࡢሙྜࠊKA31a ᰴࡢቑṪࡀほᐹࡉࢀࡓࡀࠊa924E1 ᰴ ࡢቑṪࡣほᐹ࡛ࡁ࡞ࡗࡓࠋࡇࡢ⤖ᯝࡣࠊࡇࢀࡲ࡛ࡢ Shigematsu ࡽ(2010a)ࡼ 56 ࡿ a924E1 ᰴࡀ KA31a ᰴẚ㍑ࡋ࡚ࡼࡾ㧗࠸ᅽຊឤཷᛶ⬟ࢆ᭷ࡍࡿ࠸࠺ሗ࿌ ࢆᨭᣢࡋࡓࠋࡋࡋ࡞ࡀࡽࠊࣝࢠࢽࣥࢆῧຍࡋ࡚ᇵ㣴ࡋࡓሙྜࠊ㧗ᅽฎ⌮ᚋ a924E1 ᰴ࠾ࡼࡧ KA31a ᰴࡢ୧ᰴ࠾࠸࡚ቑṪࢆほᐹࡋࡓ(ᅗ 8)ࠋࡇࢀࡽࡢ⤖ᯝ ࡣࠊࣝࢠࢽࣥࡀ㓝ẕ⣽⬊ᑐࡋ࡚ࠊ㧗ᅽ⪏ᛶᐤࡋ࡚࠸ࡿࡇࢆ♧၀ࡋ࡚࠸ ࡿࠋ ᮏ❶ࡢ⤖ᯝࢆࡲࡵࡿࠊࣝࢠࢽࣥ⏕ྜᡂ⤒㊰ࡢάᛶࡢపୗ㉳ᅉࡍࡿ ࣝࢠࢽࣥࡢᯤῬࡀࠊa924E1 ᰴࡢᅽຊឤཷᛶ⬟ࢆࡍࡿ୍ᅉ࡛࠶ࡿࡇࡀ♧၀ ࡉࢀࡓ(ᅗ 7)ࠋࡲࡓࠊᮏ❶ࡢᡂᯝࡣࣝࢠࢽࣥࡀ㓟ࡸ⤖ࢫࢺࣞࢫ⪏ᛶࡢࡳ࡞ ࡽࡎࠊ㧗ᅽࢫࢺࣞࢫ⪏ᛶ࠾࠸࡚ࡶᐤࡍࡿࡇࢆ♧၀ࡍࡿึࡵ࡚ࡢሗ࿌࡛࠶ ࡿࠋࡇࡢ᪂ࡋ࠸▱ぢࡣ PReF ᢏ⾡ࡢ㛤Ⓨࡁࡃᐤࡍࡿࡇࡔࢁ࠺ࠋ 57 ➨ 5 ❶ ⤖ㄽ ᅽຊࡣࠊ㠀ඹ᭷⤖ྜࡢࡳస⏝ࡋࠊ㠀⇕ⓗࢱࣥࣃࢡ㉁ࢆኚᛶࡉࡏࡿࡇࡼ ࡾࠊᚤ⏕≀ࡢቑṪ㜼ᐖࡸάᛶࢆᘬࡁ㉳ࡇࡍ≀⌮ⓗࢫࢺࣞࢫ࡛࠶ࡿࠋ1987 ᖺ 㧗ᅽࢆ㣗ရຍᕤᛂ⏝ࡍࡿ㧗ᅽ㣗ရຍᕤࡢᴫᛕࡀᥦၐࡉࢀࡓࠋ㣗ရ୰ࡢ᭷⏝ ᡂศࡸ᪂㩭࡞㢼ࠊⰍࠊࢃ࠸ࢆಖᣢࡋࡘࡘࠊᚤ⏕≀ࢆάᛶࡉࡏࡿࡇࡀྍ ⬟࡞ࡗࡓࠋࡋࡋࠊᚤ⏕≀ࡢάᛶࡣ 300 MPa ௨ୖࡢ㧗ᅽࡀᚲせ࡛࠶ࡿ ࡓࡵࠊ㧗ᅽ⪏࠼ࡽࢀࡿᅽຊᐜჾࡸᅽຊ⨨ࡢタഛࢥࢫࢺࡢቑ➼ࡼࡾ㧗ᅽ ᢏ⾡ࡢᬑཬࡀጉࡆࡽࢀ࡚࠸ࡿࡢࡀ⌧≧࡛࠶ࡿࠋࡑࡢၥ㢟ࢆゎỴࡍࡿࡓࡵࠊ㧗 ᅽ࣭㧗ࢥࢫࢺࡀᚲせ࡞ᚤ⏕≀ࡢ⁛⳦(sterilization)࡛ࡣ࡞ࡃࠊ100~200 MP ⛬ᗘࡢ୰ 㧗ᅽ᮲௳ࡼࡗ࡚Ⓨ㓝㣗ရࢆ⏕⏘ࡍࡿ㓝ẕࡢẅ⳦(pasteurization)↔Ⅼࢆᙜ࡚ࠊ ᅽຊࡼࡾⓎ㓝ࢆไᚚࡍࡿᢏ⾡(Pressure Regulated Fermentation; PReF)ࡀᥦࡉ ࢀࡓ(Nomura and Iwahahsi, 2014)ࠋPReF ᢏ⾡ࡢ☜❧ࡣࠊ୰㧗ᅽ᮲௳࡛άᛶ ࡍࡿᅽຊᙅ࠸㓝ẕᰴࡢసฟࡀᚲせ࡛࠶ࡿࠋ ࡇࢀࡲ࡛ᐇ㦂ᐊ㓝ẕᰴ S. cerevisiae KA31a ᰴᑐࡋ࡚⣸እ⥺↷ᑕἲࡼࡿࣛ ࣥࢲ࣒✺↛ኚ␗ࡢᑟධࡼࡗ࡚ KA31a ᰴࡼࡾࡶ㧗࠸ᅽຊឤཷᛶ⬟ࢆ♧ࡍᅽຊឤ ཷᛶኚ␗ᰴ a924E1 ᰴࢆྲྀᚓࡋࡓࠋa924E1 ᰴࡣࠊᅽຊឤཷᛶ⬟ࡢࡳ࡞ࡽࡎࠊKA31a ᰴྠ➼ࡢ࢚ࢱࣀ࣮ࣝⓎ㓝⬟ࢆ᭷ࡋ࡚࠾ࡾࠊPReF ᢏ⾡ࡢ☜❧ࡢࡓࡵࡣࡇࢀࡽ 58 ࡢ⾲⌧ᙧ㉁ࢆ᭷ࡍࡿ⏘ᴗ⏝㓝ẕᰴࡢసฟࡀᚲせ࡛࠶ࡿࠋࡋࡋࠊa924E1 ᰴࡣࣛ ࣥࢲ࣒✺↛ኚ␗ࡼࡾྲྀᚓࡉࢀࡓࡓࡵࠊᅽຊឤཷᛶኚ␗㑇ఏᏊࡀᮍࡔ᫂ࡽ ࡞ࡗ࡚࠸࡞࠸ࠋᮏ◊✲࡛ࡣࠊa924E1 ᰴࡢᅽຊឤཷᛶኚ␗㑇ఏᏊࡢྠᐃ࠾ࡼࡧࡑ ࡢᅽຊឤཷᛶᶵᵓࡢゎ᫂ࢆ┠ⓗࡋࡓࠋ➨ 2 ❶࠾ࡼࡧ➨ 3 ❶࡛ࡣࠊDNA ࣐ࢡ ࣟࣞࡼࡿ㑇ఏᏊⓎ⌧ࣉࣟࣇࣝࡢ⥙⨶ⓗゎᯒࡼࡾࠊᅽຊឤཷᛶ⬟ࢆ ࡍࡿኚ␗㑇ఏᏊࢆ⤠ࡾ㎸ࡳࠊ࣑ࢺࢥࣥࢻࣜࡢ⾲⌧ᙧ㉁ゎᯒ࠾ࡼࡧ PCR ࡼࡾᅽຊឤཷᛶኚ␗㑇ఏᏊࢆྠᐃࡋࡓࠋ➨ 4 ❶࡛ࡣࠊ࣓ࢱ࣑࣎ࣟࢡࢫࡼࡾ௦ ㅰ⏘≀ࡢࣉࣟࣇࣝࢆ⥙⨶ⓗゎᯒࡋࠊCOX1 㑇ఏᏊࡢḞኻࡼࡗ࡚ᅽຊឤཷ ᛶ⬟ࡀࡉࢀࡿ࣓࢝ࢽࢬ࣒ࢆゎᯒࡋࡓࠋ௨ୗྛ❶ࢆ⥲ᣓࡍࡿࠋ ➨ 2 ❶࡛ࡣࠊa924E1 ᰴࡢᅽຊឤཷᛶ⬟ࢆࡍࡿ㑇ఏⓗせᅉࢆゎᯒࡍࡿࡓࡵ ࠊDNA ࣐ࢡࣟࣞࡼࡾ㑇ఏᏊⓎ⌧ࣉࣟࣇࣝࢆ KA31a ᰴࡢࡶࡢẚ ㍑ゎᯒࡋࡓ(⾲ 3-6)ࠋࡑࡢ⤖ᯝࠊ5,821 㑇ఏᏊࡢⓎ⌧ࢹ࣮ࢱࢆྲྀᚓࡋࠊࡇࢀࡽࡢ㑇 ఏᏊࡢ࠺ࡕࠊa924E1 ᰴࡢ 498 㑇ఏᏊࡢⓎ⌧ࣞ࣋ࣝࡣ KA31a ᰴࡢ㑇ఏᏊࡢⓎ⌧ࣞ ࣋ࣝࡼࡾࡶ᭷ព㧗ࡃ(p < 0.05)ࠊ649 㑇ఏᏊࡢⓎ⌧ࣞ࣋ࣝࡣ᭷ពపࡗࡓ(p < 0.05)ࠋa924E1 ᰴ࠾࠸࡚ࠊ“Energy”ᶵ⬟㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇 ఏᏊ࠾ࡼࡧ“Mitochondria”ᒁᅾࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢⓎ⌧ࡀ᭱ ࡶ᭷ពࢵࣉࣞࢠ࣮ࣗࣞࢺ(p < 0.05)ࡋࡓࡇࡀ᫂ࡽ࡞ࡗࡓࠋ≉ COX1ࠊ 59 AI1ࠊCOX17ࠊCOX18ࠊCYC7ࠊPET191ࠊSHY1ࠊNCA2ࠊCOQ8ࠊMRPL1ࠊMRPS35ࠊ MBA1 㑇ఏᏊ➼ࡢ࣑ࢺࢥࣥࢻࣜࡸ࢚ࢿࣝࢠ࣮௦ㅰ㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ ࣮ࢻࡍࡿ㑇ఏᏊࡢⓎ⌧ࡀࢵࣉࣞࢠ࣮ࣗࣞࢺࡋࡓࠋࡲࡓࠊRPS3ࠊRPS5ࠊRPS31ࠊ RPL10ࠊRPL30 㑇ఏᏊ➼ࡢ͆Protein synthesis”ᶵ⬟ࡸࣂࣜࣥࠊࣟࢩࣥࠊࢯࣟ ࢩࣥࠊࣝࢠࢽࣥࠊࢭࣜࣥࠊࢺࣜࣉࢺࣇࣥ➼ࡢ࣑ࣀ㓟ࡢ⏕ྜᡂ㛵㐃ࡍࡿࢱ ࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡀ᭷ពࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺ(p < 0.05)ࡋࡓࡇࡀ ᫂ࡽ࡞ࡗࡓࠋQuantitative PCR ࡼࡗ࡚ DNA ࣐ࢡࣟࣞࡢ㑇ఏᏊⓎ⌧ ࢆホ౯ࡋࡓࡇࢁࠊa924E1 ᰴࡢࢵࣉࣞࢠ࣮ࣗࣞࢺࡋࡓ㑇ఏᏊࡢ┦ᑐⓎ⌧ࣞ࣋ ࣝࡣ KA31a ᰴࡢ┦ᑐⓎ⌧ࣞ࣋ࣝẚ㍑ࡋ࡚ 2 ಸ௨ୖࡢ್ࢆ♧ࡋࡓࠋa924E1 ᰴࡢ ࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺࡋࡓ㑇ఏᏊࡢ┦ᑐⓎ⌧ࣞ࣋ࣝࡣ KA31a ᰴࡢ┦ᑐⓎ⌧ࣞ࣋ࣝ ࡢ༙ศ௨ୗ࡛࠶ࡗࡓ(⾲ 7)ࠋࡇࢀࡽࡢ⤖ᯝࡽࠊᅽຊឤཷᛶኚ␗ࡢᑟධࡼࡾ࣑ ࢺࢥࣥࢻࣜᶵ⬟ᙳ㡪ࡀ⏕ࡌࠊࢱࣥࣃࢡ㉁ࡸ࣑ࣀ㓟ࡢ௦ㅰᶵ⬟ࡀపୗࡋ࡚ ࠸ࡿྍ⬟ᛶࢆ♧ࡋ࡚࠸ࡿࠋࡋࡋ a924E1 ᰴ࠾࠸࡚ࠊࢵࣉࣞࢠ࣮ࣗࣞࢺࡋ࡚ ࠸ࡓ COX1 㑇ఏᏊࡢⓎ⌧ࡀእⓗ☜ㄆ࡛ࡁ࡞ࡗࡓ(⾲ 7)ࠋCOX1 㑇ఏᏊࡢ ࡢ㡿ᇦࢆࢱ࣮ࢤࢵࢺࡋࡓ quantitative PCR ࡼࡾゎᯒࡋࡓ⤖ᯝ࠾࠸࡚ࡶⓎ ⌧ࡣほᐹ࡛ࡁࡎࠊࡲࡓ COX1 㑇ఏᏊࡢࣉ࣮ࣟࣈ㓄ิࡀ PRS2 㑇ఏᏊ㧗࠸┦ྠᛶ ࢆ♧ࡋࡓࡇࡽࠊa924E1 ᰴࡢ COX1 㑇ఏᏊࡢⓎ⌧ࡢぢࡅୖࡢࢵࣉࣞࢠࣗ ࣮ࣞࢺࡣࢡࣟࢫࣁࣈࣜࢲࢮ࣮ࢩ࡛ࣙࣥ࠶ࡿࡇࡀ♧၀ࡉࢀࡓࠋ 60 ➨ 3 ❶࡛ࡣࠊ㑇ఏᏊⓎ⌧ࣉࣟࣇࣝࡢ⥙⨶ⓗゎᯒࡽ᫂ࡽ࡞ࡗࡓ a924E1 ᰴࡢ࣑ࢺࢥࣥࢻࣜᶵ⬟ᅽຊឤཷᛶࡢ㛵㐃ᛶࡘ࠸࡚ゎᯒࡋࡓࠋTTC ᰁⰍ ࡼࡾ࣑ࢺࢥࣥࢻࣜࡢ྾ᶵ⬟ࢆホ౯ࡋࡓࡇࢁࠊa924E1 ᰴࡢ྾ᶵ⬟ࡢపୗ ࡀ᫂ࡽ࡞ࡗࡓ(ᅗ 1)ࠋ㓝ẕࡢ྾ᶵ⬟ࡢపୗࡣ࣑ࢺࢥࣥࢻࣜ DNA ࡢḞᦆ ㉳ᅉࡍࡿࡇࡀከ࠸ࠋ࣑ࢺࢥࣥࢻࣜ DNA ࢆ PCR ࡼࡾゎᯒࡋࡓࡇࢁࠊ COX1 㑇ఏᏊ㡿ᇦࡢḞኻࡀ᫂ࡽ࡞ࡗࡓ(ᅗ 5)ࠋࡲࡓࠊCOX1 㑇ఏᏊ௨እࡢ࣑ࢺ ࢥࣥࢻࣜ㑇ఏᏊࡢḞኻࡣ☜ㄆࡉࢀ࡞ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝࡣࠊa924E1 ᰴࡢ࣑ ࢺࢥࣥࢻࣜ྾ᶵ⬟ࡢపୗࡀ COX1 㑇ఏᏊࡢࡳࡢḞኻ㉳ᅉࡋ࡚࠸ࡿࡇࢆ ♧ࡋ࡚࠸ࡿࠋ୍⯡ⓗ࡞྾Ḟᦆ㓝ẕᰴࡣ࡚ࡢ࣑ࢺࢥࣥࢻࣜ DNA ࢆḞᦆࡋ࡚ ࠸ࡿࡀࠊ a924E1 ᰴࡣ COX1 㑇ఏᏊࡢࡳࢆḞኻࡋࡓ㠀ᖖ⛥࡞྾Ḟᦆᰴ࡛࠶ࡾࠊ ࣑ࢺࢥࣥࢻࣜ྾ᶵ⬟㛵ࡍࡿ◊✲࠾࠸࡚ࡶ᭷┈࡞ኚ␗ᰴ࡛࠶ࡿࡇࡀ ᫂ࡽ࡞ࡗࡓࠋCOX1 㑇ఏᏊࢆḞኻࡋࡓ a924E1 ᰴ(a ᆺ୍ಸయ)࠾ࡼࡧ COX1 㑇 ఏᏊࢆḞኻࡋ࡚࠸࡞࠸㔝⏕ᆺᰴ KA31α ᰴ(α ᆺ୍ಸయ)ࢆ᥋ྜࡋࠊಸయᰴࢆస ฟࡋࡓࠋࡑࡢ⤖ᯝࠊCOX1 㑇ఏᏊࡢḞኻࡣ࣑ࢺࢥࣥࢻࣜඹ⣽⬊㉁㑇ఏࡋࡓ (ᅗ 1, 5)ࠋCOX1 㑇ఏᏊḞኻ࣑ࢺࢥࣥࢻࣜࢆᣢࡘಸయᰴࡣࠊCOX1 㑇ఏᏊࢆḞ ኻࡋ࡚࠸࡞࠸㔝⏕ᆺ࣑ࢺࢥࣥࢻࣜࢆᣢࡘಸయᰴࡼࡾࡶ᭷ព㧗࠸ᅽຊឤཷ ᛶ⬟ࢆ♧ࡋࠊࡑࡢಸయᰴࡢᅽຊឤཷᛶ⬟ࡣ a924E1 ᰴ༉ᩛࡍࡿࡇࡀ᫂ࡽ 61 ࡞ࡗࡓ(ᅗ 4)ࠋࡇࢀࡽࡢ⤖ᯝࡽࠊ࣑ࢺࢥࣥࢻࣜ DNA ࡢ COX1 㑇ఏᏊࡢḞ ኻࡀᅽຊឤཷᛶᙉࡃ㛵㐃ࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋ ➨ 4 ❶࡛ࡣࠊ࣓ࢱ࣑࣎ࣟࢡࢫࡼࡾ௦ㅰ⏘≀ࡢ⥙⨶ⓗゎᯒࡼࡾࠊCOX1 㑇ఏ ᏊࡢḞኻࡼࡿᅽຊឤཷᛶ⬟ࡢᶵᵓࢆゎᯒࡋࡓ(⾲ 8)ࠋࡑࡢ⤖ᯝࠊCE/MS ゎ ᯒࡼࡾࠊ250 ✀㢮ࡢྜ≀ࢆ᳨ฟࡋࠊa924E1 ᰴ࠾࠸࡚ 48 ✀㢮ࡢྜ≀ࡀ KA31a ᰴẚ㍑ࡋ࡚┦ᑐⓗῶᑡࡋࡓ(p < 0.05)ࠋࡲࡓࠊ59 ✀㢮ࡢྜ≀ࡣࠊ a924E1 ᰴ࡛ࡣ᳨ฟࡉࢀࡎࠊα-ࢣࢺࢢࣝࢱࣝ㓟➼ࡢ࠸ࡃࡘࡢྜ≀ࡣ a924E1 ᰴ ࠾ࡼࡧ KA31a ᰴࡢ୧ᰴ᳨࡛ฟࡉࢀ࡞ࡗࡓࠋࡇࢀࡽࡢ┦ᑐⓗῶᑡࡋࡓྜ≀ ࠶ࡿ࠸ࡣ a924E1 ᰴ᳨࡛ฟࡉࢀ࡞ࡗࡓྜ≀ࡣ TCA ࢧࢡࣝࡸࣝࢠࢽࣥ⏕ ྜᡂ㛵ࡋ࡚࠸ࡓ(⾲ 9, ᅗ 7)ࠋDNA ࣐ࢡࣟࣞࡼࡿ㑇ఏᏊⓎ⌧ࡢ⥙⨶ ⓗゎᯒࡢ⤖ᯝࡽࠊa924E1 ᰴ࠾࠸࡚ࠊࡇࢀࡽࡢྜ≀ࡢ௦ㅰ㛵㐃ࡍࡿ㓝⣲ ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊ(CIT2ࠊCIT3ࠊGDH1ࠊGDH3ࠊIDP2ࠊSDH1ࠊSDH2ࠊSHH3ࠊ ARG1ࠊARG3ࠊARG4ࠊARG7ࠊCPA1ࠊMAK31ࠊPUT1ࠊVBA1 㑇ఏᏊ)ࡢⓎ⌧ࡀࢲ ࢘ࣥࣞࢠ࣮ࣗࣞࢺࡋࡓࡇࡀ᫂ࡽ࡞ࡗ࡚࠸ࡿࠋᵝࠎ࡞ࢫࢺࣞࢫ⪏ᛶᐤ ࡍࡿࣝࢠࢽࣥ⏕ྜᡂ㛵ࡍࡿ㓝⣲ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊ࡛࠶ࡿ ARG8ࠊARG3ࠊ ARG1ࠊARG4 㑇ఏᏊࡢⓎ⌧ࢆ RT-PCR ࡛ゎᯒࡋࡓࡇࢁࠊa924E1 ᰴ࠾࠸࡚Ⓨ ⌧ࡢపୗࡀ᫂ࡽ࡞ࡗࡓ(ᅗ 6)ࠋࣝࢠࢽࣥࡀᅽຊ⪏ᛶᐤࡍࡿᙳ㡪ࢆホ౯ 62 ࡍࡿࡓࡵࠊࣝࢠࢽࣥῧຍࡲࡓࡣᮍῧຍࡢ᮲௳࡛ᇵ㣴ࡋࡓᚋ㧗ᅽฎ⌮ࢆ ࡋࡓࠋࣝࢠࢽࣥᮍῧຍࡢሙྜࠊKA31a ᰴࡢቑṪࡀほᐹࡉࢀࡓࡀࠊa924E1 ᰴࡢ ቑṪࡣほᐹ࡛ࡁ࡞ࡗࡓ(ᅗ 8)ࠋࡇࡢ⤖ᯝࡣࠊࡇࢀࡲ࡛ࡢ a924E1 ᰴࡀ KA31a ᰴ ẚ㍑ࡋ࡚ࡼࡾ㧗࠸ᅽຊឤཷᛶ⬟ࢆ᭷ࡍࡿ࠸࠺ሗ࿌ࢆᨭᣢࡋࡓ(Shigematsu et al., 2010a)ࠋࡲࡓࠊ10 mM ௨ୖࡢࣝࢠࢽࣥࢆῧຍࡋ࡚ᇵ㣴ࡋࡓሙྜࠊa924E1 ᰴ ࡢᅽຊ⪏ᛶࡢྥୖࡀ☜ㄆࡉࢀࡓ(ᅗ 8)ࠋࡇࢀࡽࡢ⤖ᯝࡽࠊࣝࢠࢽࣥࡀ㓝ẕ⣽ ⬊ᑐࡋ࡚㧗ᅽ⪏ᛶᐤࡋ࡚࠸ࡿࡇࢆ᫂ࡽࡋࡓࠋ ௨ୖࠊᮏ◊✲࠾࠸࡚ࠊ࣑ࢺࢥࣥࢻࣜ DNA ࡢ COX1 㑇ఏᏊࡀᅽຊ⪏ᛶᐤ ࡍࡿࡇࡀ᫂ࡽ࡞ࡗࡓࠋᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴࡣࠊCOX1 㑇ఏᏊࡢ Ḟኻࡼࡿ࣑ࢺࢥࣥࢻࣜᶵ⬟ࡢపୗࡼࡾࠊTCA ࢧࢡࣝࡸࣝࢠࢽࣥ⏕ྜ ᡂ㛵㐃ࡍࡿ௦ㅰᶵ⬟ࡀపୗࡋࡓࡇࡀ♧ࡉࢀࡓ(ᅗ 7)ࠋᮏ◊✲࡛ࡣࠊࡇࢀࡽࡢ ⤖ᯝࡽᘬࡁ㉳ࡇࡉࢀࡓࣝࢠࢽࣥࡢᯤῬࡀᅽຊឤཷᛶ⬟ࢆࡋࡓ୍ᅉ࡛࠶ ࡿࡇࢆ᫂ࡽࡋࡓࠋࡲࡓࠊᮏ◊✲ࡣࠊ㓟ࡸ⤖ࢫࢺࣞࢫ⪏ᛶᐤࡍࡿ ࣝࢠࢽࣥࡀࠊ㧗ᅽࢫࢺࣞࢫ⪏ᛶᑐࡋ࡚ࡶᐤࡍࡿࡇࢆ♧ࡍึࡵ࡚ࡢሗ࿌࡛ ࠶ࡿࠋᮏ◊✲ࡢᡂᯝࡣࠊCOX1 㑇ఏᏊḞኻ࣑ࢺࢥࣥࢻࣜࢆ⏘ᴗ⏝㓝ẕᰴ౪ ࡍࡿࡇ࡛ࠊᅽຊឤཷᛶ⏘ᴗ⏝㓝ẕᰴࢆᐜ᫆సฟࡍࡿࡇࡀྍ⬟࡛࠶ࡿࡇ ࢆ♧၀ࡋࠊPReF ᢏ⾡ࡢ☜❧ࡢࡓࡵࡢ㊊ࡾ࡞ࡿࡇࡀᮇᚅ࡛ࡁࡿࠋ 63 ➨ 6 ❶ ㅰ㎡ ᒱ㜧Ꮫ ᒾᶫ ᆒ ᩍᤵࡣࠊᮏ◊✲ࡢ⯡࠾ࡼࡧᮏ༤ኈㄽᩥࡢᇳ➹࠾ࡁࡲ ࡋ࡚ࡈᣦᑟࢆ㈷ࡾࡲࡋࡓࠋ◊✲ᑐࡍࡿ⪃࠼᪉ࠊㄽᩥࡸ⏦ㄳ᭩ࡢᵓᡂࠊᐇ⏝◊ ✲࠾ࡅࡿࢥࢫࢺព㆑➼ࠊ⊂≉ࡢ࣮ࣘࣔࢆ࠼࡚ ࡃࡈᣦᑟ࠸ࡓࡔࡁࡲࡋ ࡓࠋࡲࡓࠊࢼࣀ⢏Ꮚࡸື≀ᐇ㦂➼ࡢ㧗ᅽ◊✲௨እࡢ◊✲ࠊᏛࡢ㐠Ⴀࡢ⿵ຓ➼ࢆ ㏻ࡌ࡚࡛ࡣ⤒㦂ࡍࡿࡇࡀ㞴ࡋ࠸ࡇࢆᏛࡪᶵࢆ࠼࡚࠸ࡓࡔࡁࡲࡋࡓࠋ ᚰࡼࡾᚚ♩⏦ࡋୖࡆࡲࡍࠋ࠶ࡾࡀ࠺ࡈࡊ࠸ࡲࡋࡓࠋ ᒱ㜧Ꮫ 㕥ᮌ ᚭ ᩍᤵࡣࠊᮏ◊✲ࡢ㐙⾜ࡸ୰㛫Ⓨ⾲࡚ࡈຓゝ࠸ࡓࡔࡁࠊ ᮏ༤ኈㄽᩥࡢᵓᡂ࠾ࡼࡧᑂᰝ➼࠾ࡁࡲࡋ࡚ࡈᣦᑟࢆ㈷ࡾࡲࡋࡓࠋࡲࡓࠊㄽᩥࡢ సᡂࡸࢻࢡࢱ࣮ࢥ࣮ࢫࡢྜᐟ➼ࡢᵝࠎ࣋ࣥࢺ࡛ ࡃࡈᣦᑟ࠸ࡓࡔࡁࡲࡋࡓࠋ ཌࡃᚚ♩⏦ࡋୖࡆࡲࡍࠋ 㟼ᒸᏛ ᑠᕝ ┤ே ᩍᤵࡣࠊᮏ◊✲ࡢ㐙⾜ࡸ୰㛫Ⓨ⾲࡚ࡈຓゝ࠸ࡓࡔࡁࠊ ᮏ༤ኈㄽᩥࡢᵓᡂ࠾ࡼࡧᑂᰝ➼࠾ࡁࡲࡋ࡚ࡈᣦᑟࢆ㈷ࡾࡲࡋࡓࠋࡲࡓࠊ◊✲ࡢ 㐍ࡵ᪉ࡸᐇ㦂ࡢᡭᢏࠊᡭἲࡘࡁࡲࡋ࡚ ࡃࡈᣦᑟ࠸ࡓࡔࡁࡲࡋࡓࠋཌࡃᚚ♩ ⏦ࡋୖࡆࡲࡍࠋ ᪂₲⸆⛉Ꮫ 㔜ᯇ ᩍᤵࠊཱྀ ᚨ ຓᩍࡣࠊᮏ◊✲࡛⏝࠸ࡓ S. cerevisiae KA31a ᰴ࠾ࡼࡧ a924E1 ᰴࠊࡑࢀࡽࡢಸయᰴ➼ࢆศࡋ࡚࠸ࡓࡔࡁ 64 ࡲࡋࡓࠋࡲࡓࠊᮏ◊✲ࡢ㐙⾜࠾ࡼࡧㄽᩥࡢᇳ➹࠾ࡁࡲࡋ࡚㐺ษ࡞ࡈຓゝࢆ㈷ࡾ ࡲࡋࡓࠋཌࡃᚚ♩⏦ࡋୖࡆࡲࡍࠋ ⏘ᴗᢏ⾡⥲ྜ◊✲ᡤࡢ㧗ᶫ ῟Ꮚ ༤ኈࠊ㣗ရ⥲ྜ◊✲ᡤࡢ㕥ᮌ ᛅᏹ ༤ኈ ࡣࠊDNA ࣐ࢡࣟࣞࡢゎᯒࡘࡁࡲࡋ࡚ࡈᣦᑟࢆ㈷ࡾࡲࡋࡓࠋ῝ࡃᚚ♩⏦ ࡋୖࡆࡲࡍࠋ ᒱ㜧Ꮫ ୰ᕝ ᬛ⾜ ᩍᤵࠊ୰ᮧ ᾈᖹ ᩍᤵࡣࠊ࣑ࢺࢥࣥࢻࣜࡢᰁⰍᐇ 㦂࠾ࡼࡧ⺯ග㢧ᚤ㙾ࡢほᐹ࠾ࡁࡲࡋ࡚ࡈᣦᑟࢆ㈷ࡾࡲࡋࡓࠋ῝ࡃᚚ♩⏦ࡋୖ ࡆࡲࡍࠋ ᒱ㜧Ꮫ ᒾ㛫 ᬛᚨ ᩍᤵࡣࠊ◊✲ࡢ㐙⾜࠾ࡼࡧ◊✲⏕ά࠾ࡁࡲࡋ࡚㐺 ษ࡞ࡈຓゝࢆ㈷ࡾࡲࡋࡓࠋ῝ࡃᚚ♩⏦ࡋୖࡆࡲࡍࠋ ࠾ྡ๓ࢆ࠾ᣲࡆ࡛ࡁࡲࡏࢇ࡛ࡋࡓࡀࠊᏛ➼࡛ᮏ◊✲ࡘࡁࡲࡋ࡚ከࡃࡢඛ ⏕᪉ࡈຓゝࢆ㈷ࡾࡲࡋࡓࠋ῝ࡃᚚ♩⏦ࡋୖࡆࡲࡍࠋ ᭱ᚋࠊ㛗࠸Ꮫ⏕⏕άࢆᨭ࠼࡚࠸ࡓࡔ࠸ࡓ୧ぶ♽∗ẕࠊᘵឤㅰ࠸ࡓࡋࡲࡍࠋ ࠶ࡾࡀ࠺ࡈࡊ࠸ࡲࡋࡓࠋ 65 ➨ 7 ❶ ཧ⪃ᩥ⊩ [1] Abe, F., and Kato, C. (1999) Barophysiology. In: Horikoshi, K., and Tsuji, K. [eds.] Extremophile in Deep-sea Environments, pp. 227-248. Springer, Berlin. [2] Adegoke, G. O., and Babalola, A. K. (1988) Characteristics of microorganisms of importance in the fermentation of fufu and ogi ̺ two Nigerian foods. J. Appl. Bacteriol. 65, 449-453. [3] Aertsen, A., Houdt, R. V., Vanoirbeek, K., and Michiels, C. W. (2004) An SOS response induced by high pressure in Escherichia coli. J. Bacteriol. 186, 6133-6141. [4] Basak, S., Ramaswamy, H. S., and Piette, J. P. G. (2002) High pressure destruction kinetics of Leuconostoc mesenteroides and Saccharomyces cerevisiae in single strength and concentrated orange juice. Innov. Food Sci. Emerg. Technol. 3, 223-231. [5] Brauer, M. J., Saldanha, A. J., Dolinski, K., and Botstein, D. (2005) Homeostatic adjustment and metabolic remodeling in glucose-limited yeast cultures. Mol. Biol. Cell 16, 2503–17. [6] Bravim, F., Lippman S. I., Silva, L. F. D., Souza, D. T., Fernandes, A. A. R., Masuda, C. A., Broach, J. R., Fernandes, P. M. B. (2012) High hydrostatic pressure activates gene expression that leads to ethanol production enhancement in a Saccharomyces 66 cerevisiae distillery strain. Appl. Microbiol. Biotechnol. 97, 2093-2107. [7] Bridgman, P. W. (1912) Water, in the liquid and five solid forms, under pressure. Proc. Amer. Acad. Arts Sci. 47, 439-558. [8] Bridgman, P. W. (1914) The coagulation of albumen by pressure. J. Biol. Chem. 19, 511-512. [9] Chong, P. L., Fortes, P. A., and Jameson, D. M. (1985) Mechanisms of inhibition of (Na, K)-ATPase by hydrostatic pressure studied with fluorescent probes. J. Biol. Chem. 260, 14484-14490. [10] Egilmez, N. K., and Jazwinski, S.M. (1989) Evidence for the involvement of a cytoplasmic factor in the aging of the yeast Saccharomyces cerevisiae. J. Bacteriol. 171, 37-42. [11] Fernandes, P. M., Domitrovic, T., Kao, C. M., Kurtenbach, E. (2004) Genomic expression pattern in Saccharomyces cerevisiae cells in response to high hydrostatic pressure. FEBS Lett. 556, 153–6. [12] Freitas, J. M. D., Bravim, F., Buss, D. S., Lemos, E. M., Fernandes, A. A. R., and Fernandes, P. M. B. (2012) Influence of cellular fatty acid composition on the response of Saccharomyces cerevisiae to hydrostatic pressure stress. FEMS Yeast Res. 12, 871-878. 67 [13] ⚟ཎ ᏹ (1986) 㓝ẕ࣑ࢺࢥࣥࢻࣜ㑇ఏᏊࡢᵓ㐀ᶵ⬟. Ꮫ⏕≀, 24, 707-716. [14] ⚟ᓮ ⱥ୍㑻 (2013) ࣓ࢱ࣑࣎ࣟࢡࢫࡢ⌧≧ྍ⬟ᛶ, ⚟ᓮ ⱥ୍㑻[eds.] ࣓ ࢱ࣑࣎ࣟࢡࢫࡢ᭱ඛ➃ᢏ⾡ᛂ⏝<ᬑཬ∧>, ➨ 1 ∧, 1-10. ࢩ࣮࢚࣒ࢩ࣮ฟ ∧, ᮾி. [15] Galloway, J. N., Townsend, A. R., Erisman, J. W., Bekunda, M., Cai, Z., Freney, J. R., Martinelli, L. A., Seitzinger, S. P., and Sutton, M. A. (2008) Transformation of the Nitrogen Cycle: Recent Trends, Questions, and Potential Solutions. Science 320, 889-892. [16] Goffeau, A., Barrell, B. G., Bussey, H., Davis, R. W., Dujon, B., Feldmann, H., Galibert, F., Hoheisel, J. D., Jacq, C., Johnston, M., Louis, E. J., Mewes, H. W., Murakami, Y., Philippsen, P., Tettelin, H., and Oliver, S. G. (1996) Life with 6000 Genes. Science 274, 546-567. [17] Hamada, H., and Shiraki, K. (2007) L-Argininamide improves the refolding more effectively than L-arginine. J. Biotechnol. 130, 153-160. [18] Haney, S. A., Xu, J., Lee, S. Y., Ma, C. L., Duzic, E., Broach, J., and Manfredi, J. (2001) Genetic selection in Saccharomyces of mutant mammalian adenylyl cyclases with elevated basal activities Mol. Gen. Genet. 265, 1120-1128. 68 [19] Hartwell, L. H. (2004) Yeast and Cancer. Biosci. Rep. 24, 523-544. [20] Hasegawa, T., Hayashi, M., Nomura, K., Hayashi, M., Kido, M., Ohmori, T., Fukuda, M., Iguchi, A., Ueno, S., Shigematsu, T., Hirayama, M., and Fujii, T. (2012) Highthroughput method for kinetic analysis on high pressure inactivation of microorganisms using microplate. J. Biosci. Bioeng. 113, 788–91. [21] Hashizume, C., Kimura, K., and Hayashi, R. (1995) Kinetic Analysis of Yeast Inactivation by High Pressure Treatment at Low Temperatures. Biosci. Biotech. Biochem. 59, 1455-1458. [22] Hayashi, R., Kawamura, Y., and Kunugi, S. (1987) Introduction of High Pressure to Food Processing: Preferential Proteolysis of β-Lactoglobulin in Milk Whey. J. Food Sci. 52: 1107–1108. [23] ᯘ ຊ (1991) 㣗ရࡢᅽຊຍᕤ㸫ຍᕤ㣗ရࡢ≀ᛶ. ⇕≀ᛶ 5, 284-290. [24] ᯘ ຊ (2008) ⏕≀࠼ࡿᅽຊຠᯝຍᅽ㣗ရࡢᑠྐ. 㧗ᅽຊࡢ⛉Ꮫ ᢏ⾡ 18, 70-377. [25] Hite, B. (1899) The effect of pressure in the preservation of milk. Bull. W. VA U. Agricultural Experiment Station 58: 15-35. [26] Hite, B., Giddings, N. J., and Weakley, Jr. C. E. (1914) The effect of pressure on certain microorganisms encountered in the preservation of fruits and veges. Bull. W. 69 VA U. Agricultural Experiment Station. 146: 1-67. [27] Hohmann, S. (2002) Osmotic Stress Signaling and Osmoadaptation in Yeasts. Microbiol. Mol. Biolo. Rev. 66, 300-372. [28] ᇼỤ 㞝, ᮌᮧ 㑥⏨, ⏣ 㞞ኵ (1991) ຍᅽἲࡼࡿࢪ࣒ࣕ〇㐀㛵ࡍࡿ◊ ✲. ᪥ᮏ㎰ⱁᏛㄅ 65, 975-980. [29] Ishii, S. (2002) Some microbiological findings on kumiss and their functions. J. Brewing Society of Japan 97, 210-215. [30] Iwahashi, H., Kaul, S. C., Obuchi, K., and Komatsu, Y. (1991) Induction of barotolerance by heat shock treatment in yeast. FEMS Microbiol. Lett. 80, 325-328. [31] ᒾᶫ ᆒ (2002) DNA ࣐ࢡࣟࣞ, DNA ࢳࢵࣉ㸫ࡇࢀࡽࡢ᪂ᢏ⾡㸫. ⎔ ቃᢏ⾡ 31, 679-685. [32] Iwahashi, H., Shimizu, H., Odani, M., and Komatsu, Y. (2003) Piezophysiology of genome wide gene expression levels in the yeast Saccharomyces cerevisiae. Extremophiles 7, 291–98. [33] Iwahashi, H., Odani, M., Ishidou, E., and Kitagawa, E. (2005) Adaptation of Saccharomyces cerevisiae to high hydrostatic pressure causing growth inhibition. FEBS Lett. 579, 2847-52. [34] Iwahashi, H., Kitagawa, E., Suzuki, Y., Ueda, Y., Ishizawa, Y., Nobumasa, H., 70 Kuboki, Y., Hosoda, H., and Iwahashi, Y. (2007) Evaluation of toxicity of the mycotoxin citrinin using yeast ORF DNA microarray and Oligo DNA microarray. BMC Genomics 8, 1-13. [35] Iwahashi, Y., Kitagawa, E., and Iwahashi, H. (2008) Analysis of mechanisms of T-2 toxin toxicity using yeast DNA microarrays. Int. J. Mol. Sci. 9, 2585-2600. [36] Jaenicke, L. (2007) Centenary of the award of a Nobel Prize to Eduard Buchner, the father of biochemistry in a test tube and thus of experimental molecular bioscience. Angew. Chem. Int. Edit. 46: 6776-6782. [37] Kasuga, K. (1998) Outline of the results of the food industry extrahigh-pressure application technology research association. Food and Technology 320: 15-17. [38] Kawarai, T., Arai, S., Furukawa, S., Ogihara, H., and Yamasaki, M. (2006) Highhydrostatic- pressure treatment impairs actin cables and budding in Saccharomyces cerevisiae. J. Biosci. Bioeng. 101, 515-518. [39] ᮌᮧ 㐍 (1993) 㣗ရ⏘ᴗࡢᮍ᮶ࢆᣅࡃ㸫㧗ᅽᢏ⾡㧗ᐦᗘᇵ㣴. 㣗ရ⏘ᴗ ㉸㧗ᅽ⏝ᢏ⾡◊✲⤌ྜ[Eds.] ᗣ⏘ᴗ᪂⪺, ᮾி. [40] Kobori, H., Sato, M., Tameike, A., Hamada, K., Shimada, S., Osumi, M. (1995) Ultrastructural effects of pressure stress to the nucleus in Saccharomyces cerevisiae: a study by immunoelectron microscopy using frozen thin sections. FEMS Microbiol. 71 Lett. 132, 253. [41] Koseki, S., and Yamamoto, K. (2006) Recovery of Escherichia coli ATCC 25922 in phosphate buffered saline after treatment with high hydrostatic pressure. Int. J. Food Microbiol. 110, 108-111. [42] Kurita, S., Kitagawa, E., Kim, C. H., Momose, Y., and Iwahashi, H. (2002) Studies on the antimicrobial mechanisms of capsaicin using yeast DNA microarray. Bios. Biot. Biochem. 66: 532-536. [43] Lee, J.W., Cha, D.S., Park, H.J., and Hwang, K.T. (2003) Effects of CO2 absorbent and high-pressure treatment on the shelf-life of packaged Kimchi products. Int. J. Food Sci. Technol. 38, 519-524. [44] Liti, G., Carter, D. M., Moses, A.M., Warringer, J., Parts, L., James, S. A., Davey, R. P., Roberts, I. N., Burt, A., Koufopanou, V., Tsai, I. J., Bergman, C. M., Bensasson, D., O’Kelly, M. J. T., Oudenaarden, A. V., Barton, D. B. H., Bailes, E., Nguyen, A. N., Jones, M., Quail, M. A., Goodhead, I., Sims, S., Smith, F., Blomberg, A., Durbin, R., and Louis, E. J. (2009) Population genomics of domestic and wild yeasts. Nature 458, 337-341. [45] Lyutova, E. M., Kasakov, A. S., and Gurvits, B.Y. (2007) Effects of Arginine on Kinetics of Protein Aggregation Studied by Dynamic Laser Light Scattering and 72 Tubidimetry Techniques. Biotechnol. Prog. 23, 1411-1416. [46] Matsuoka, H., Suzuki, Y., Iwahashi, H., Arao, T., Suzuki, Y., and Tamura, K. (2005) The biological effects of high-pressure gas on the yeast transcriptome. Braz. J. Med. Biol. Res. 38, 1267-1272. [47] Mitsuoka, T., Makita, S., and Asano, H. (2002) The characteristics of lactic acid bacteria isolated from Masai fermented milk in Kenya. Biosci. Microflora 21, 171178. [48] Mohammed, S. I., Steenson, L. S., and Kireleis, A. W. (1991) Isolation and characterization of microorganisms associated with the traditional sorghum fermentation for production of Sudanese kisra. Appl. Environ. Microbiol. 57, 25292533. [49] Momose, Y., and Iwahashi, H. (2001) Bioassay of cadmium using DNA microarray: Genome-wide expression patterns of Saccharomyces cerevisiae response to cadmium. Env. Tox. Chem. 20: 2353-2360. [50] Morita, Y., Nakamori, S., and Takagi, H. (2002) Effect of proline and arginine metabolism on freezing stress of Saccharomyces cerevisiae. J. Biosci. Bioeng. 94, 390-394. [51] Murata, Y., Watanabe, T., Sato, M., Momose, Y., Nakahara, T., Oka, S., and Iwahashi, 73 H. (2003) DMSO exposure facilitates phospholipid biosynthesis and cellular membrane proliferation in yeast cells. J. Biol. Chem. 278: 33185-33193. [52] Murokoshi, A. (2004) Development of a device for shucking oysters. Nippon Suisan Gakk., 70: 671-673. [53] Nagai, S. (1959) Induction of the respiration-deficient mutation in yeast by various synthetic dyes. Science 130, 1188–1189. [54] Nishimura, A., Kotani, T., Sasano, Y., and Takagi, H. (2010) An antioxidative mechanism mediated by the yeast N-acetyltransferase Mpr1: oxidative stress-induced arginine synthesis and its physiological role. FEMS Yeast Res. 10, 687-698. [55] 㔝ᮧ ୍ᶞ, ᒾᶫ ᆒ (2013) 㧗ᅽୗ࠾ࡅࡿ㓝ẕ⣽⬊ࡢṚ. 㧗ᅽຊࡢ⛉Ꮫ ᢏ⾡ 23, 53-58. [56] Nomura, K., and Iwahashi, H. (2014) Pressure-Regulated Fermentation: A revolutionary approach that utilizes hydrostatic pressure. Reviews in Agricultural Science 2, 1-10. [57] Odani, M., Komatsu, Y., Oka, S., and Iwahashi, H. (2003) Screening of genes that respond to cryopreservation stress using yeast DNA microarray. Cryobiol. 47: 155164. [58] ᳃ ୣ, 㔜ஂ ಖ (1990) ␆⏘ཬࡰࡍ㧗ᅽస⏝. In: ᯘ ຊ[eds.] ຍᅽ㣗 74 ရ, ➨ 1 ∧ pp. 131-139. ࡉࢇ࠼࠸ฟ∧, ி㒔. [59] Ohshima, S., Nomura, K., and Iwahashi, H. (2013) Clarification of the recovery mechanism of Escherichia coli after hydrostatic pressure treatment. High Pressure Res. 33, 308-314. [60] Okada, S., Ishikawa, M., Yoshida, I., Uchimura, T., Ohara, N., and Kozaki, M. (1992) Identification and characteristics of lactic acid bacteria isolated from sour dough sponges. Biosci. Biotech. Biochem. 56, 572-575. [61] Oyewole, O. B., and Odunfa, S. A. (1988) Microbiological studies on cassava fermentation for ‘lafun’ production. Food Microbiol. 5, 125-133. [62] Pascal, B. (1653) ᯇᾉಙ୕㑻ヂ (1953) ࣃࢫ࢝ࣝ⛉Ꮫㄽᩥ㞟, ➨ 4 ∧. Pp. 1203. ᒾἼ᭩ᗑ, ᮾி. [63] Patrignani, F., Vannini, L., Kamdem, S. L. S., Lanciotti, L., and Guerzoni, M. E. (2009) Effect of high pressure homogenization on Saccharomyces cerevisiae inactivation and physico-chemical features in apricot and carrot juices. Int. J. Food Microbiol. 136, 23-31. [64] Pope, D. H., and Berger, L. R. (1973) Inhibition of metabolism by hydrostatic pressure: What limits microbial growth? Arch. Microbiol. 93, 367–370. [65] Roger, H. (1892) Action des hautes pressions sur quelques bactéries. C. R. Hebd. 75 Acad. Sci. 114. [66] Roger, H. (1895) Action des hautes pressions sur quelques bactéries. Arch. Physiol. Norm. Path. 7:12–17. [67] Shigematsu, T., Nasuhara, Y., Nagai, G., Nomura, K., Ikarashi, K., Hayashi, M., Ueno, S., and Fujii, T. (2010a) Isolation and characterization of barosensitive mutants of Saccharomyces cerevisiae obtained by UV mutagenesis. J. Food Sci. 75, M509M514. [68] Shigematsu, T., Nomura, K., Nasuhara, Y., Ikarashi, K., Nagai, G., Hirayama, M., Hayashi, M., Ueno, S., and Fujii, T. (2010b) Thermosensitivity of a barosensitive Saccharomyces cerevisiae mutant obtained by UV mutagenesis. High Pressure Res. 30, 524-529. [69] 㔜ᯇ (2013) 㧗ᅽຊୗ⌧㇟ࡢ≉ᚩᏛᛂ. In: 㔜ᯇ , すᾏ ⌮அ [eds.] 㐍ࡍࡿ㣗ရ㧗ᅽຍᕤᢏ⾡㸫ᇶ♏ࡽ᭱᪂ࡢᛂ⏝ࡲ࡛㸫, ➨ 1 ∧ pp. 11-16. ࢚ࢾ࣭ࢸ࣮࣭࢚ࢫ, ᮾி. [70] Sirisattha, S., Momose, Y., Kitagawa, K. and Iwahashi, H. (2004) Genomic profile of Roundup treatment of yeast using DNA microarray analysis. Environ. Sci. 11: 313323. [71] 㕥ᮌ ᩔኈ (2011) ᪂₲┴ࡀ࣮ࣜࢻࡍࡿ㣗ရࡢ㧗ᅽຍᕤฎ⌮ᢏ⾡. New 76 Food Ibdustry 53, 47-56. [72] 㕥ᮌ ᩔኈ (2013) 㣗ရຍᕤ࠾ࡅࡿඛ➃ຍᕤᢏ⾡㧗ᅽᢏ⾡ࡢ᭱᪂. In: 㔜ᯇ , すᾏ ⌮அ[eds.] 㐍ࡍࡿ㣗ရ㧗ᅽຍᕤᢏ⾡㸫ᇶ♏ࡽ᭱᪂ࡢ ᛂ⏝ࡲ࡛㸫, ➨ 1 ∧ pp. 11-16. ࢚ࢾ࣭ࢸ࣮࣭࢚ࢫ, ᮾி. [73] Tamura, K., Shimizu, T., and Kourai, H. (1992) Effects of ethanol on the growth and elongation of Escherichia coli under high pressure up to 40 MPa. FEMS Microbiol. Lett. 99, 321-324. [74] Tanaka, Y., Higashi, T., Rakwal, R., Wakida, S., and Iwahashi, H. (2007) J. Pharm. Biomed. Anal. 44, 608-613. [75] ⏣୰ ႐⚽, ᮾ ဴྖ, Rakwal, R., ᰘ⸨ ῟Ꮚ, ⬥⏣ ៅ୍, ᒾᶫ ᆒ (2013) ࢤ ࣀ࣑ࢡࢫ࣓ࢱ࣑࣎ࣟࢡࢫࡢ⏕≀Ꮫⓗゎᯒᢏ⾡ࡋ࡚ࡢ⼥ྜ, ⚟ᓮ ⱥ୍㑻 [eds.] ࣓ࢱ࣑࣎ࣟࢡࢫࡢ᭱ඛ➃ᢏ⾡ᛂ⏝<ᬑཬ∧>, ➨ 1 ∧, pp. 195-203. ࢩ ࣮࢚࣒ࢩ࣮ฟ∧, ᮾி. [76] ㎷⏣⣧, 㕥ᮌ㑥ᏹ (1990) 㧗ᅽฎ⌮ᢏ⾡ࢆ⏝ࡋࡓ㣗⫗ຍᕤရ. In: ᯘ ຊ [eds.] ຍᅽ㣗ရ, ➨ 1 ∧ pp. 123-130. ࡉࢇ࠼࠸ฟ∧, ி㒔. [77] Tsumoto, K., Umetsu, M., Kumagai, I., Ejima, D., Philo, J. S., and Arakawa, T. (2004) Role of Arginine in Protein Refolding, Solubilization, and Purification. Biotechnol. Prog. 20, 1301-1308. 77 [78] ㉸㧗ᅽᢏ⾡ࡢ㣗ရ➼ࡢᛂ⏝㛵ࡍࡿ◊✲ (1991) 㧗ᅽ⏝㛵ࡍࡿ◊✲ ᡂᯝሗ࿌᭩. pp. 1-88. ⴥ༳ๅ, ᪂₲. [79] Vogel, R. F., Pavlovic, M., Hörmann, S., and Ehrmann, M. A. (2005) High pressuresensitive gene expression in Lactobacillus sanfranciscensis. Braz. J. Med. Biol. Res. 38, 1247-1252. [80] ᒣᮏ ㈗, ᑠ㛵 ᡂᶞ (2009) 㣗ရ㧗ᅽຍᕤᢏ⾡. 㣗⣊㸫ࡑࡢ⛉Ꮫᢏ⾡㸫 47, 37-50. [81] ᒣᓮ ᙯ, ➲ᕝ ⛅ᙪ (2000) 㧗ᅽࢆ⏝ࡋࡓ⡿ຍᕤ㣗ရࡢ㛤Ⓨ. ᪥ᮏ㎰ⱁ Ꮫㄅ 74, 619-623. [82] Yasokawa, D., Murata, S., Kitagawa, E., Iwahashi, Y., Nakagawa, R., Hashido, T., and Iwahashi, H. (2008) Mechanisms of copper toxicity in Saccharomyces cerevisiae determined by microarray analysis. Environ Toxicol. 23, 599-606. [83] Yasokawa, D., Murata, S., Iwahashi, Y., Kitagawa, E., Kishi, K., Okumura, Y., and Iwahashi, H. (2010) DNA microarray analysis suggests that zinc pyrithione causes iron starvation to the yeast Saccharomyces cerevisiae. J. Biosci. Bioeng. 109, 479486. [84] Yasokawa, D. and Iwahashi, H. (2010) Toxicogenomics using yeast DNA microarrays. J. Biosci. Bioeng. 110, 511-522. 78 [85] Yayanos, A. A., and Pollard, E. C. (1969) A study of the effects of hydrostatic pressure on macromolecular synthesis in Escherichia coli. Biophys. J. 9, 1464–82. [86] ZoBell, C. E. and Cobet, A. B. (1964) Filament formation by Escherichia coli at increased hydrostatic pressures. J. Bacteriol. 87, 710-719. 79 ➨ 8 ❶ ᅗ⾲ Table 1. Saccharomyces cerevisiae strains used in this study. rho+; normal respiration strain, rho-; respiration-deficient strain. Strain Genotype Saccharomyces cerevisiae KA31a MATa his3 leu2 trp1 ura3 Saccharomyces cerevisiae a924E1 MATa his3 leu2 trp1 ura3 Saccharomyces cerevisiae KA31α MATα his3 leu2 trp1 ura3 Parent rho+ type haploid Barosensitive mutant rho㸫 type haploid Wild type rho+ type haploid Saccharomyces cerevisiae C208 MATa/α his3 leu2 trp1 ura3 rho㸫 type diploid Saccharomyces cerevisiae E201 MATa/α his3 leu2 trp1 ura3 rho+ type diploid Saccharomyces cerevisiae E208 MATa/α his3 leu2 trp1 ura3 rho㸫 type diploid Saccharomyces cerevisiae F102 MATa/α his3 leu2 trp1 ura3 rho㸫 type diploid Saccharomyces cerevisiae F108 MATa/α his3 leu2 trp1 ura3 rho+ type diploid 80 5’-ATGGGTGAAACCATTCCATT-3’ 5’-ACAGATTCCCCTTGACCTTG-3’ 5’-CGTCCTGAGGATGAGTCTCA-3’ 5’-TTGATCGTTTTTCCCACAAA-3’ 5’-GGTTGGCAGTGTTCATCAAC-3’ 5’-TCACCGAAGCTGTTCTTGTC-3’ 5’-TTTAGTGGTATGGCAGGAACAG-3’ 5’-GCTACAGATACAGCATTTCCAAGA-3’ 5’-GAAAAGGGGGTGGGAGTAAA-3’ 5’-CGCACTTTGCAGAAACGATA-3’ 5’-ATGCGGGAAAACCCTAAAGT-3’ 5’-TCTTTGCTGGTTTATTCTGAGC-3’ 5’-GGGTTCTTTGATGCTGATGG-3’ 5’-TCCAAGCACATCAAGAAACC-3' 5’-TGTCAACCACAGCATCCAC-3’ 5’-GCCTTTGGATGTTTTCTTGG-3’ 5’-ACTATGGGGTGGGAGATTCA-3’ 5’-ATTGCCGAAAGAATGCAAAAGG-3’ Aldehyde dehydrogenase Catabolism of arginine Mitochondrial ribosomal protein Fructose-1,6-bisphosphatase Isocitrate lyase Succinate-fumarate carrier Cytochrome c oxidase Cytochrome c oxidase 15S ribosomal RNA 21S ribosomal RNA Cytochrome c oxidase Cytochrome c oxidase Cytochrome B Cell division cycle N-acetyl-ornithine aminotransferase Ornithine carbamoyltransferase Arginisuccinate synthetase Argiosuccinate lyase Actin ALD3 CAR1 MRP51 FBP1 ICL1 SFC1 COX1 COX1 15S rRNA 21S rRNA COX1 COX3 COB CDC48 ARG8 ARG3 ARG1 ARG4 ACT1 81 5’-ACCAAAAAGGACGAGATTGC-3’ F-primer Gene function Gene name Table 2. Sequences of oligonucleotide primers used in this study. 5’-CGCACAAAAGCAGAGATTAGAAACA-3’ 5’-CGTAAGGATGACCAGCAAGA-3’ 5’-TCTGGGGTGAAGTAGGAACC-3’ 5’-TCACACGGGCAAAAATACAT-3’ 5’-ACTCAGCACCAAGCATCAAA-3’ 5’-CCTCCACAAACAATCTCGTG-3’ 5’-TATGGGAGTTCCCACAAAGC-3’ 5’-TACCTGCGATTAAGGCATGA-3’ 5’-CCCTGTACCAGCACCTGATT-3’ 5’-ATAATGACGCCCCATCAAAA-3’ 5’-GATTCGCATGTGTCATGTCC-3’ 5’-CACCACCTCCTGATACTTCAAA-3’ 5’-CGTAGTAAGTATCGTGGAATGCTA-3’ 5’-TTTGGCTTTGGTGTGTCATT-3’ 5’-CTAGCCATTGGTCAGGGAAT-3’ 5‘-ATCCGTACATGGCATAGCAA-3’ 5’-CCAGCCATTTATTGTCGTTG-3’ 5’-ACACCATCGACGTCATAGGA-3’ 5’-CCATGTGGGTTCCTAAAGCT-3’ R-primer Control Downregulated Downregulated Downregulated Downregulated Control Mitochondria Mitochondria Mitochondria Mitochondria Mitochondria COX1 gene 2 COX1 gene 1 Downregulated Downregulated Downregulated Upregulated Upregulated Upregulated Table 3. Functional categories of upregulated genes in the mutant strain a924E1. p-values indicate the statistical significance of the observed upregulation of genes in each category. Number of Number of Percentage of Total genes Altered genes Altered genes Energy 367 45 12.3% 0.003 Biogenesis of cellular components 862 88 10.2% 0.012 Unclassified proteins 1393 128 9.2% 0.042 Metabolism 1514 137 9.0% 0.085 Protein synthesis 480 42 8.8% 0.341 Cell fate 273 22 8.1% 1.000 Interaction with the environment 463 37 8.0% 1.000 Protein fate 1154 91 7.9% 1.000 Cellular transport 1038 76 7.3% 1.000 Cell type differentiation 452 33 7.3% 1.000 Cell cycle and DNA processing 1012 73 7.2% 1.000 Regulation of metabolism 253 17 6.7% 1.000 Binding proteins 1049 66 6.3% 1.000 Cell rescue, defense and virulence 554 34 6.1% 1.000 Signal transduction mechanism 234 13 5.6% 1.000 Transcription 1077 57 5.3% 1.000 Development 69 3 4.3% 1.000 Transposable elements 120 3 2.5% 1.000 Functional Category 82 P value Table 4. Locational categories of upregulated genes in the mutant strain a924E1. p-values indicate the statistical significance of the observed upregulation of genes in each category. Number of Number of Percentage of Total genes Altered genes Altered genes 1047 124 11.8% 0.001> Cell wall 44 10 22.7% 0.002 Extracellular 54 11 20.4% 0.004 Bud 150 17 11.3% 0.104 Cell periphery 216 23 10.6% 0.113 Endosome 58 7 12.1% 0.192 Cytoskeleton 204 20 9.8% 0.226 Microsomes 5 1 20.0% 0.347 Ambiguous 237 21 8.9% 0.381 172 15 8.7% 0.436 Plasma membrane 186 15 8.1% 0.454 Transport vesicles 141 11 7.8% 1.000 ER 552 40 7.2% 1.000 Punctate composite 140 10 7.1% 1.000 Cytoplasm 2844 190 6.7% 1.000 Vacuole 284 19 6.7% 1.000 Golgi 158 10 6.3% 1.000 Nucleus 2136 131 6.1% 1.000 Peroxisome 52 2 3.8% 1.000 Lipid particles 27 1 3.7% 1.000 Locational Category Mitochondria Integral membrane / endomembranes 83 P value Table 5. Functional categories of downregulated genes in the mutant strain a924E1. p-values indicate the statistical significance of the observed downregulation of genes in each category. Functional Category Number of Number of Total genes Altered genes Percentage of Altered genes P value Protein synthesis 480 155 32.3% 0.001> Transcription 1077 155 14.4% 0.001> Binding proteins 1049 148 14.1% 0.001> Energy 367 48 13.1% 0.071 Metabolism 1514 174 11.5% 0.111 69 8 11.6% 0.453 Cell rescue, defense and virulence 554 57 10.3% 1.000 Cell fate 273 24 8.8% 1.000 Cellular transport 1038 90 8.7% 1.000 Cell cycle and DNA processing 1012 83 8.2% 1.000 Protein fate 1154 85 7.4% 1.000 Cell type differentiation 452 30 6.6% 1.000 Regulation of metabolism 253 16 6.3% 1.000 Biogenesis of cellular components 862 52 6.0% 1.000 Interaction with the environment 463 27 5.8% 1.000 Signal transduction mechanism 234 13 5.6% 1.000 Unclassified proteins 1393 74 5.3% 1.000 Transposable elements 120 5 4.2% 1.000 Development 84 Table 6. Locational categories of downregulated genes in the mutant strain a924E1. p-values indicate the statistical significance of the observed downregulation of genes in each category. Number of Number of Percentage of Total genes Altered genes Altered genes Cytoplasm 2844 386 13.6% 0.001> Nucleus 2136 270 12.6% 0.001> Peroxisome 52 7 13.5% 0.313 Microsomes 5 1 20.0% 0.430 Plasma membrane 186 16 8.6% 1.000 Vacuole 284 24 8.5% 1.000 ER 552 46 8.3% 1.000 Mitochondria 1047 86 8.2% 1.000 Cell periphery 216 17 7.9% 1.000 Lipid particles 27 2 7.4% 1.000 172 11 6.4% 1.000 Golgi 158 8 5.1% 1.000 Cell wall 44 2 4.5% 1.000 Bud 150 6 4.0% 1.000 Ambiguous 237 9 3.8% 1.000 Extracellular 54 2 3.7% 1.000 Punctate composite 140 5 3.6% 1.000 Cytoskeleton 204 7 3.4% 1.000 Transport vesicles 141 4 2.8% 1.000 Locational Category Integral membrane / endomembranes 85 P value Table 7. Confirmation of DNA microarray analysis results. ALD3, CAR1, and MRP51 genes of the mutant strain were upregulated compared to those of the parent strain, and FBP1, ICL1, and SFC1 genes were downregulated. Relative expression level of the upregulated genes of the mutant strain was higher than that of the parent strain, and relative expression level of the downregulated genes was lower. Relative expression level of the COX1 gene was very low or it could not be detected. The CDC48 gene was amplified as a control. Reproducibility was confirmed by performing at least 3 independent experiments. N.D.; not detected. *; p < 0.05, **; p < 0.01. Gene Relative expression level name KA31a a924E1 ALD3 1.00 4.82 ± 1.09 * Upregulated gene CAR1 1.00 2.70 ± 0.59 * Upregulated gene MRP51 1.00 2.57 ± 0.62 * Upregulated gene FBP1 1.00 0.03 ± 0.03 ** Downregulated gene ICL1 1.00 0.12 ± 0.13 ** Downregulated gene SFC1 1.00 0.02 ± 0.02 ** Downregulated gene COX1 1.00 < 0.01 ** COX1 gene 1 COX1 1.00 N.D. COX1 gene 2 CDC48 1.00 1.12 ± 0.53 Control 86 Table 8. Total detected metabolites. Metabolomics was carried out with three independent experiments (*; p < 0.05, **; p < 0.01, ***; p < 0.001). N.A.; Not Available. Compound name Fold P-value㻌㻌 ATP 0.01 0.109 Gln 0.02 0.009 Guanidoacetic acid 0.02 GDP-glucose Compound name Fold P-value Sarcosine 0.19 N.A. ** Mevalolactone 0.19 0.013 * 0.012 * p-Aminobenzoic acid 0.19 0.010 ** 0.03 0.037 * Imidazolelactic acid 0.20 0.001> *** UDP-N-acetylglucosamine 0.06 0.061 2-Hydroxyvaleric acid 0.20 0.069 N-Acetylleucine 0.06 0.015 N-Acetylornithine 0.20 0.001> *** Butyric acid 0.07 0.090 3-Hydroxypropionic acid 0.20 0.001> *** Citric acid 0.07 0.051 Isovaleric acid 0.21 0.096 3-Hydroxy-3-methylglutaric acid 0.07 0.076 Thiamine diphosphate 0.21 0.054 Mevalonic acid 0.07 0.071 Tyr 0.21 0.008 Creatinine 0.08 0.007 ** CMP-N-acetylneuraminate 0.21 0.066 Anthranilic acid 0.09 0.013 * Acetoacetamide 0.21 0.006 ** 2-Isopropylmalic acid 0.09 0.074 3-Methylhistidine 0.21 0.006 ** Asp 0.09 0.012 * Glutathione (GSSG)_divalent 0.22 0.028 * Uridine 0.11 0.013 * Glu 0.22 0.005 ** Fructose 6-phosphate 0.11 0.051 Phosphoenolpyruvic acid 0.22 0.007 ** Sucrose 6'-phosphate 0.11 0.012 * Hydroxyproline 0.24 0.013 * Pro 0.11 0.010 ** O-Acetylcarnitine 0.25 0.009 ** Leu 0.12 0.005 ** N-Acetylasparagine 0.26 0.018 * Argininosuccinic acid 0.13 0.016 * Carnosine 0.26 0.011 * Betaine 0.13 0.002 ** 2-Methylserine 0.28 0.004 ** Phosphorylcholine 0.14 0.019 * Val 0.28 0.006 ** Ophthalmic acid 0.14 0.055 N6-Acetyllysine 0.28 0.022 * 2-Hydroxy-4-methylvaleric acid 0.15 0.043 * N-Acetylhistidine 0.30 0.044 * 2-Aminoisobutyric acid 0.15 0.002 ** Cystine 0.33 0.066 3-Hydroxybutyric acid 0.15 0.012 * N-Acetyllysine 0.33 0.150 Malic acid 0.15 0.059 Cysteine glutathione disulfide 0.33 0.074 1-Methyladenosine 0.15 0.014 * 2-Aminoadipic acid 0.33 0.105 Adenine 0.16 0.021 * Carboxymethyllysine 0.33 0.049 cis-4-Hydroxyproline 0.16 0.006 ** N-Ethylglycine 0.33 0.001> *** Isobutyrylcarnitine 0.16 0.042 * His 0.36 0.001> *** Ile 0.17 0.001> *** Ornithine 0.37 0.030 N-Acetylglutamic acid 0.18 0.067 Octanoic acid 0.37 N.A. * 87 ** * * Compound name Fold P-value㻌㻌 Compound name Fold P-value Isovalerylcarnitine 0.37 0.013 * SDMA 0.66 0.136 Nicotinamide 0.38 0.045 * Heptanoic acid 0.67 0.166 S-Methylcysteine 0.39 0.003 ** Citrulline 0.67 0.114 N-Acetylglycine 0.40 0.023 * Thiamine 0.67 0.017 Gluconic acid 0.40 0.022 * S-Adenosylmethionine 0.69 0.080 Creatine 0.40 0.003 ** FAD_divalent 0.72 0.398 5'-Deoxy-5'-methylthioadenosine 0.40 0.006 ** Lys 0.73 0.016 4-Guanidinobutyric acid 0.41 0.054 Trp 0.73 0.063 N-Methylalanine 0.42 N.A. Carbachol 0.74 0.539 Isoniazid 0.43 N.A. Thr 0.75 0.082 Glycerophosphocholine 0.43 0.001> *** Thiamine phosphate 0.76 0.549 Arg 0.44 0.001> *** Ala 0.77 0.085 Trimethylamine 0.45 N.A. Glyceric acid 0.78 0.254 Pyridoxamine 0.47 0.205 p-Toluic acid 0.78 0.441 Dyphylline 0.47 0.009 Pelargonic acid 0.78 0.204 Glutathione (GSH) 0.48 0.615 Pyridoxamine 5'-phosphate 0.79 0.158 O-Acetylhomoserine 0.48 N.A. γ-Butyrobetaine 0.81 0.008 NAD+ 0.48 0.057 4-Methyl-5-thiazoleethanol 0.84 0.270 β-Ala-Lys 0.48 0.124 NMN 0.84 0.096 N6-Methyllysine 0.49 0.001> *** Cys 0.87 0.844 Asn 0.49 0.019 * 5-Hydroxylysine 0.90 0.298 γ-Glu-2-aminobutyric acid 0.50 N.A. FMN 0.92 0.733 Theobromine 0.50 0.089 5-Oxohexanoic acid 0.93 0.737 S-Lactoylglutathione 0.51 0.011 Homoserinelactone 0.95 0.753 Ser 0.52 0.109 Imidazole-4-acetic acid 0.96 0.601 Thiaproline 0.53 0.253 N-Acetylputrescine 0.98 0.853 ADMA 0.54 0.007 GDP 0.98 0.951 Xanthine 0.56 0.064 Lauric acid 0.99 0.955 N6,N6,N6-Trimethyllysine 0.58 0.007 ** Urea 1.0 0.999 5-Aminovaleric acid 0.58 0.003 ** 11-Aminoundecanoic acid 1.0 0.916 1-Aminocyclopropane-1-carboxylic acid 0.58 0.398 Morpholine 1.0 0.930 Nω-Methylarginine 0.58 0.035 3-Aminopropane-1,2-diol 1.0 0.289 Hexanoic acid 0.59 0.223 N8-Acetylspermidine 1.1 0.798 NADP+ 0.60 0.271 NADH 1.1 0.713 Phe 0.60 0.004 ** N-Acetylglucosamine 6-phosphate 1.1 0.782 S-Methylglutathione 0.63 0.050 * Cyclohexylamine 1.2 0.694 2-Amino-2-(hydroxymethyl)-1,3-propanediol 0.64 0.439 UDP 1.2 0.611 ** * ** * 88 * * ** Compound name Fold P-value㻌㻌 Compound name Fold P-value ADP 1.2 0.585 5-Amino-4-oxovaleric acid 7.0 0.014 * Choline 1.2 0.117 Orotic acid 7.4 0.004 ** Pantothenic acid 1.2 0.281 UMP 11.1 0.011 * Decanoic acid 1.2 0.709 GMP 15.6 0.005 ** 3-Amino-2-piperidone 1.2 0.083 AMP 22.7 0.002 ** Threonic acid 1.2 0.383 S-Adenosylhomocysteine 32.2 0.011 * Glycerol 1.4 0.127 Pipecolic acid 43.9 0.001> Taurocholic acid 1.4 0.536 1-Pyrroline 5-carboxylic acid 1> N.A. Trimethylamine N-oxide 1.4 0.059 2-Hydroxyglutaric acid 1> N.A. Spermidine 1.5 0.388 2-Oxoisovaleric acid 1> N.A. Ala-Ala 1.5 0.050 2-Phosphoglyceric acid 1> N.A. Urocanic acid 1.7 0.269 3-Hydroxykynurenine 1> N.A. Lactic acid 1.8 0.104 3-Methyladenine 1> N.A. Triethanolamine 1.8 0.275 3-Phosphoglyceric acid 1> N.A. Cystathionine 1.8 0.009 ** 4-Acetamidobutanoic acid 1> N.A. Gly 1.9 0.002 ** 1> N.A. Nicotinic acid 1.9 0.008 ** 5-Oxo-2-tetrahydrofurancarboxylic acid 1> N.A. Met 2.2 0.003 ** 6-Aminohexanoic acid 1> N.A. Hypoxanthine 2.3 0.033 * Acetyl CoA_divalent 1> N.A. Carnitine 2.5 0.001> *** Acetylcholine 1> N.A. Diethanolamine 2.6 0.001> *** Arg-Glu 1> N.A. 5-Oxoproline 2.6 0.003 ** Benzoic acid 1> N.A. 2.7 N.A. Butyrylcarnitine 1> N.A. Ethanolamine 2.8 0.001 ** CDP 1> N.A. Tyramine 3.0 0.010 * cis-Aconitic acid 1> N.A. Methionine sulfoxide 3.0 0.039 * CoA_divalent 1> N.A. γ-Glu-Cys 3.0 0.197 CTP 1> N.A. CDP-choline 3.4 0.003 ** Cytidine 1> N.A. Saccharopine 3.6 0.004 ** Ectoine 1> N.A. CMP 4.3 0.006 ** Fructose 1,6-diphosphate 1> N.A. Adenosine 5.4 0.005 ** Fumaric acid 1> N.A. Cholic acid 5.9 0.005 ** Glucose 1-phosphate 1> N.A. Putrescine 6.0 0.083 Glucose 6-phosphate 1> N.A. GABA 6.5 0.001> *** Gly-Asp 1> N.A. Succinic acid 6.5 0.001> *** Glycerol 3-phosphate 1> N.A. myo-Inositol 1-phosphate myo-Inositol 3-phosphate 89 4-Methyl-2-oxovaleric acid 3-Methyl-2-oxovaleric acid *** Compound name Fold P-value㻌㻌 Compound name Fold P-value Gly-Gly 1> N.A. Propionic acid 1> N.A. GTP 1> N.A. Propionyl CoA_divalent 1> N.A. Guanosine 1> N.A. PRPP 1> N.A. His-Glu 1> N.A. Pyridoxal 1> N.A. HMG CoA_divalent 1> N.A. Pyridoxine 1> N.A. Hypotaurine 1> N.A. Ribose 5-phosphate 1> N.A. Indole-3-ethanol 1> N.A. Sedoheptulose 7-phosphate 1> N.A. Inosine 1> N.A. Trehalose 6-phosphate 1> N.A. Isobutyryl CoA_divalent 1> N.A. 1> N.A. Isocitric acid 1> N.A. Uracil 1> N.A. Kynurenine 1> N.A. UTP 1> N.A. Metronidazole 1> N.A. >1 N.A. N-Acetylglutamine 1> N.A. >1 N.A. N-Acetylphenylalanine 1> N.A. >1 N.A. N-Acetylserine 1> N.A. Histidinol >1 N.A. N-Acetyl-β-alanine 1> N.A. Homoserine >1 N.A. NADPH_divalent 1> N.A. IMP >1 N.A. N-Methylglutamic acid 1> N.A. N2-Succinylornithine >1 N.A. N-Methylproline 1> N.A. N-Acetylalanine >1 N.A. Orotidine 5'-monophosphate 1> N.A. γ-Glu-Val-Gly >1 N.A. UDP-glucose UDP-galactose 2'-Deoxyadenosine 5'-Deoxyadenosine Aminoacetone Ethanolamine phosphate 90 Table 9. Relatively decreased metabolites observed in the barosensitive mutant. Metabolites were selected by a Welch’s t-test (p < 0.05) and by a two-fold cutoff in relative values compared with the parent strain. ە ; metabolites not detected in the mutant strain but present in the parent strain. ×; metabolite not detected in either strain. Relatively decreased Compound name Fold Glutamine 0.02 Aspartate 0.09 Argininosuccinic acid 0.13 Glutamate 0.22 Ornithine 0.37 Arginine 0.44 Succinic acid 6.48 cis-Aconitic acid 䠉● Isocitric acid 䠉● Acetyl CoA divalent 䠉● Fumaric acid 䠉● 2-Oxoglutaric acid 䠉× 91 Fig. 1. Respiratory function of the mutant strains. TTC staining of the parent strain (A and B), the mutant strain (C and D), the diploid strain group with normal respiration (E and F), and the respiration-deficient diploid strain group (G and H). Before staining, colonies were white (A, C, E, and G). The parent strain and the diploid strain group with normal respiration turned red at least 100 min after staining (B and F), while the mutant strain and the respiration-deficient diploid strain group became faintly pink within the same time frame (D and H). Reproducibility was confirmed in at least 3 independent experiments. 92 Fig. 2. DNA contents and cell sizes of the diploid strains. DNA contents and cell sizes observed in a mixture of the diploid strains and the haploid strains. DNA contents of the diploid strains (G1 phase: 2C, G2 phase: 4C) were approximately 2-fold compared to those of the haploid strains (G1 phase: 1C, G2 phase: 2C), and cell sizes were also similar. Reproducibility was confirmed in at least three independent experiments. 93 Fig. 3. Growth ability of the mutant strains. Growth rates of the parent strain (open triangles), the mutant strain (open diamonds), the TTCpositive diploid strain group (shaded triangles), and the TTC-negative diploid strain group (shaded diamonds) were shown. Cell concentrations were measured by A660. 94 Fig. 4. Barosensitivity of the diploid strains. Barosensitivity of the parent strain (open triangles), the mutant strain (open diamonds), the TTC-positive diploid strain group (shaded triangles), and the TTC-negative diploid strain group (shaded diamonds) after pressurized treatment at 200 MPa for 0–360 s. Means and standard deviations are shown from at least 4 independent experiments. 95 Fig. 5. Confirmation of gene deletion of the COX1 gene. K, KA31a (parent strain); E, a924E1 (mutant strain); P, TTC-positive diploid strain group; N, TTC-negative diploid strain group. Amplifications proceeded for 25 cycles. Amplification of the CDC48 gene was used as a positive control. The results shown are representative of at least 3 independent experiments. 96 Fig. 6. Gene expression involved in arginine biosynthesis. S; Molecular weight marker, W; the parent strain, M; the mutant strain. 97 Fig. 7. Arginine biosynthesis pathway. The vertical axis represents the relative values of each metabolite in the bar graphs. The dotted squares indicate genes for which mRNAs were decreased in the mutant strain. increased compound, ە ۑ ; relatively ; compounds not detected in the mutant strain but present in the parent strain, x; compound not detected in either strain. Metabolomics were carried out with three independent experiments (*; p < 0.05, **; p < 0.01). 98 Fig. 8. Growth ability after treatment with or without arginine. Treated cells were serially diluted from 10-1 to 10-2 and 5 μL of undiluted solutions and diluted solutions were spotted onto plates (from left to right). 99
© Copyright 2024 Paperzz
