酵母Saccharomyces cerevisiaeにおける圧力感受性機構の解明

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://hdl.handle.net/20.500.12099/51019 ※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。

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Saccharomyces cerevisiae ࡟࠾ࡅࡿᅽຊឤཷᛶᶵᵓࡢゎ᫂

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Saccharomyces cerevisiae ࡟࠾ࡅࡿᅽຊឤཷᛶᶵᵓࡢゎ᫂

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➨1 ❶ ᗎㄽ ... 1 ➨1 ⠇ 㟼Ỉᅽ ... 1 ➨1 㡯 ᅽຊࡢ≉ᛶ ... 2 ➨2 㡯 㧗ᅽ࡟ࡼࡿᚤ⏕≀ࡢ୙άᛶ໬ ... 3 ➨3 㡯 㧗ᅽ◊✲ࡢṔྐ㸫኱ẼᅽࡢⓎぢ࠿ࡽ㧗ᅽ◊✲ࡢⓎᒎ㸫 ... 5 ➨4 㡯 㧗ᅽ◊✲ࡢṔྐ㸫㣗ရ࡬ࡢ㧗ᅽຍᕤࡢᛂ⏝㸫 ... 8 ➨2 ⠇ 㓝ẕ ... 12 ➨1 㡯 ࣔࢹࣝ⏕≀࡜ࡋ࡚ࡢ㓝ẕ ... 13 ➨2 㡯 Ⓨ㓝㣗ရ࡜ࡋ࡚ࡢ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

➨1 ⠇ ⥴ゝ ... 20 ➨1 㡯 DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ ... 20 ➨2 㡯 DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒࡢᛂ⏝౛ ... 21 ➨3 㡯 DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ࡟࠾ࡅࡿࢡࣟࢫࣁ࢖ࣈࣜࢲ࢖ࢮ࣮ࢩࣙࣥ ... 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 ࣐࢖ࢡࣟ࢔ࣞ࢖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Ⓨࣉࣟࣇ࢓࢖ࣝࡢᴫ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 ࣑ࢺࢥࣥࢻࣜ࢔ᶵ⬟ࡢゎᯒ ... 35 ➨1 ⠇ ⥴ゝ ... 35 ➨1 㡯 㓝ẕ࡜࣑ࢺࢥࣥࢻࣜ࢔ ... 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 ᅽຊ୙άᛶ໬ᣲືࡢゎᯒ ... 42 ➨5 㡯 㔝⏕ᆺ࣑ࢺࢥࣥࢻࣜ࢔࡟ࡼࡿḞኻ㑇ఏᏊࡢ⿵᏶ ... 42 ➨4 ⠇ ⪃ᐹ ... 44 ➨4 ❶ ࣓ࢱ࣑࣎ࣟࢡࢫ࡟ࡼࡿᅽຊឤཷᛶᶵᵓࡢ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 ࢔ࣝࢠࢽࣥࡢᅽຊ୙άᛶ໬࡬ࡢᐤ୚ ... 54 ➨4 ⠇ ⪃ᐹ ... 55 ➨5 ❶ ⤖ㄽ ... 58 ➨6 ❶ ㅰ㎡ ... 64 ➨7 ❶ ཧ⪃ᩥ⊩ ... 66 ➨8 ❶ ᅗ⾲ ... 80

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1 ❶ ᗎㄽ

➨1 ⠇ 㟼Ỉᅽ  ᅽຊ (pressure) ࡜ࡣࠊ༢఩㠃✚࠶ࡓࡾ࡟స⏝ࡍࡿຊ࡜ᐃ⩏ࡉࢀࡿ⇕ຊᏛⓗࣃ ࣓࣮ࣛࢱࡢ୍✀࡛࠶ࡿࠋᮏ◊✲࡛⏝࠸ࡿ㟼Ỉᅽ (hydrostatic pressure) ࡜ࡣࠊỈ➼ ࡢᾮయࢆ፹య࡜ࡋࡓᅽຊࡢࡇ࡜࡛࠶ࡿࠋ௨ୗࠊ㟼Ỉᅽࡣᅽຊ࡜グ㍕ࡍࡿࠋ᭱ࡶ㌟ ㏆࡟Ꮡᅾࡍࡿᅽຊࡢ౛࡜ࡋ࡚኱Ẽᅽࡀ࠶ࡿࠋᆅ⾲ࡢ኱Ẽᅽࡣ࠾ࡼࡑ 1 Ẽᅽ࡛࠶ ࡾࠊྂࡃࡣ኱Ẽࢆព࿡ࡍࡿatmosphere ࠿ࡽ 1 atm ࡜ࡶ⾲ࡉࢀࡓࠋ⌧ᅾ࡛ࡣᅜ㝿༢ ఩⣔࡟ࡼࡾ1 Ẽᅽ=1,013 hPaҸ0.1 MPa ࡜⾲♧ࡍࡿࠋ1 Pa (ࣃࢫ࢝ࣝ)ࡣࠊ1 N/m2_࡜ ᐃ⩏ࡉࢀ࡚࠸ࡿࠋ኱Ẽᅽࡣ኱Ẽࡢ㔜ࡉ࡜ࡶ⪃࠼ࡽࢀࠊ㧗ᗘࡀ㧗ࡃ࡞ࡿ࡯࡝ࠊ኱Ẽ ᅽࡣపࡃ࡞ࡿࠋࡲࡓࠊỈ୰࡛ࡣỈ῝ࡀ10 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

2 ➨1 㡯 ᅽຊࡢ≉ᛶ  ᅽຊࡣࠊ ᗘ࡜ྠᵝ࡟ࢠࣈࢬ࢚ࢿࣝࢠ࣮ࢆኚ໬ࡉࡏࡿࣃ࣓࣮ࣛࢱ࡛࠶ࡾࠊ࠶ࡿ ⣔ࡢ≧ែࢆኚ໬ࡉࡏࡿࡓࡵ࡟⾜౑࡛ࡁࡿ࢚ࢿࣝࢠ࣮࡛࠶ࡿࠋ౛࠼ࡿ࡞ࡽࡤࠊ㣗ရ ࡢຍᕤࡀࢃ࠿ࡾ᫆࠸ࠋ㣗ရࡣຍ⇕ࡍࡿࡇ࡜࡟ࡼࡾࠊ㣗ရ୰ࡢᵝࠎ࡞ศᏊ㐠ືࡀά Ⓨ࡟࡞ࡾࠊỈ➼ࡢ᥹ⓎᡂศࡀẼ໬ࡋ࡚ᾘኻࡋࠊศᏊྠኈࡢ⾪✺ᅇᩘࡀቑ࠼࡚ศᏊ 㛫ࡢ໬Ꮫ཯ᛂࡀಁ㐍ࡉࢀࡿ(ᒣᮏ, ᑠ㛵, 2009)ࠋࡑࡢ⤖ᯝࠊศᏊ୰ࡢᐁ⬟ᇶࡀ௚ࡢ ᐁ⬟ᇶ࡜཯ᛂࡋ࡚᪂ࡓ࡞ຍ⇕⏕ᡂ≀ࢆ⏕ࡌࡓࡾࠊඹ᭷⤖ྜࡀ㛤⿣ࡋ࡚᭷⏝ᡂศ ࡀኻࢃࢀࡓࡾࡍࡿࠋ୍᪉ࠊ㧗ᅽ࡟ࡼࡿຍᕤᢏ⾡ࡣࠊ✚ᴟⓗ࡞ຍ⇕ࢆకࢃ࡞࠸ᗈ⩏ ࡢ㠀⇕ⓗฎ⌮࡛࠶ࡿࡓࡵ࡟ࠊୖグࡢ⇕ฎ⌮࡛ಁ㐍ࡉࢀࡿ໬Ꮫ཯ᛂࡣཎ๎ⓗ࡟㉳ ࡇࡽ࡞࠸ࠋࡍ࡞ࢃࡕࠊ㧗ᅽୗ࡟࠾࠸࡚ࡣඹ᭷⤖ྜࡢ᪂ࡓ࡞⏕ᡂࡸ㛤⿣ࡣ㉳ࡇࡽࡎࠊ 㠀ඹ᭷⤖ྜࡢࡳࡀᙳ㡪ࢆཷࡅࡿ(ᯘ, 1991)ࠋ౛࠼ࡤࠊࢱࣥࣃࢡ㉁➼ࡢᕧ኱ศᏊ࡛ ࡣࠊࡑࡢ❧యᵓ㐀ෆࡢ✵㝽ࢆᇙࡵࡿࡼ࠺࡟ࠊࡑࢀࡲ࡛ศᏊࢆᏳᐃ໬ࡋ࡚࠸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␗࡞ࡿࠋࡇࡢࡇ࡜ࡣຍ⇕࡛ࡣᘬࡁ㉳ࡇࡏ࡞࠸⌧㇟ ࢆຍᅽ࡟ࡼࡾᘬࡁ㉳ࡇࡍࡇ࡜ࢆྍ⬟࡟ࡋࠊࡲࡓࡑࡢ㏫ࡶ㉳ࡇࡾᚓࡿࡇ࡜ࢆ♧ࡋ ࡚࠸ࡿࠋ

3

➨2 㡯 㧗ᅽ࡟ࡼࡿᚤ⏕≀ࡢ୙άᛶ໬

ᅽຊࡣࠊ㠀ඹ᭷⤖ྜ࡟స⏝ࡋ࡚㠀⇕ⓗ࡟ࢱࣥࣃࢡ㉁ࢆኚᛶࡉࡏࡿࠋࡑࡢࡓࡵ࡟

㧗ᅽࡣ⏕≀࡟ᑐࡍࡿ≀⌮ⓗࢫࢺࣞࢫ࡛࠶ࡿ࡜ゝ࠼ࡿࠋࡇࢀࡲ࡛࡟㧗ᅽ࡟ࡼࡿᚤ

⏕≀ࡢቑṪ㜼ᐖࡸ୙άᛶ໬࡟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୚࠼ࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Nomura et al., 2014)ࠋࡲࡓࠊ40 MPa ⛬ᗘࡢ㧗 ᅽ᮲௳࡛ࡣࠊ4°C ࡛ࡣ⮴Ṛⓗ࡞ᦆയࢆཷࡅࡿࡀࠊ25°C ࡛ࡣቑṪࡍࡿࡇ࡜ࡀྍ⬟ ࡛࠶ࡿ(Iwahashi et al., 2003)ࠋHashizume ࡽ(1995)ࡣ 120~300 MPaࠊ-20~50°Cࠊ2~40 min ࡢ㧗ᅽຊ᮲௳࡛㓝ẕࡢ୙άᛶ໬ࢆ㏿ᗘㄽⓗ࡟ゎᯒࡋࡓࠋࡑࡢ⤖ᯝࠊ180 MPa ௨ୗࡢ㧗ᅽຊ⠊ᅖ࡟࠾࠸࡚ࠊ0~40°C ࡢ ᗘ⠊ᅖ࡛ࡣ࡯࡜ࢇ࡝୙άᛶ໬ࡀㄆࡵࡽ ࢀ࡞࠿ࡗࡓࡀࠊ-10°C ௨ୗ࠾ࡼࡧ 50°C ௨ୖࡢ ᗘᇦ࡛ࡣྠᅽຊ࡛኱ࡁ࡞୙άᛶ ໬ࡀㄆࡵࡽࢀࡓࠋ≉࡟-20°C ࡟࠾ࡅࡿ୙άᛶ໬ຠᯝࡀ㢧ⴭ࡛࠶ࡾࠊప ᇦ࡛ࡢ㧗 ᅽຊຠᯝࡢ᭷ຠᛶࡀ♧ࡉࢀࡓࠋ㧗ᅽ᮲௳ୗ࡛ࡣỈࡣịⅬୗ࡛࠶ࡗ࡚ࡶᾮయࡢ≧ ែ࡛Ꮡᅾࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿࠋ㧗ᅽ࠾ࡼࡧప ࡢ᮲௳ࡣ࡝ࡕࡽࡶศᏊ㐠ືࢆ పୗࡉࡏࠊ⣽⬊⭷ࡢὶືᛶࢆపୗࡉࡏࡿࡼ࠺࡟ᶵ⬟ࡍࡿࠋ⣽⬊⭷ࡢὶືᛶࡢపୗ ࡣࠊࣜࣥ⬡㉁➼ࡢ⭷ࢱࣥࣃࢡ㉁ࡢ┦㌿⛣࡟ࡼࡾᘬࡁ㉳ࡇࡉࢀࡿ(Chong et al., 1985)ࠋ

4

ࡑࡢ⤖ᯝࠊ⭷ᵓ㐀ࡀ◚ቯࡉࢀࠊ⣽⬊ࡀ୙άᛶ໬ࡍࡿ࡜⪃࠼ࡽࢀ࡚࠸ࡿ(Freitas et

al., 2012)ࠋ

Escherichia coli ⣽⬊࡟㧗ᅽฎ⌮ࢆ᪋ࡋ࡚୙άᛶ໬ࢆ☜ㄆࡋࡓᚋ࡟ࠊࣜࣥ㓟ࣂࢵ

ࣇ࢓࣮(PBS)୰࡛ 25°Cࠊ1 week ᇵ㣴ࡍࡿ࡜ࠊࡑࢀࡽࡢ⣽⬊ࡀᅇ᚟ࡍࡿ࡜࠸࠺⌧㇟ ࡀሗ࿌ࡉࢀࡓ(Koseki and Yamamoto, 2006)ࠋ୙άᛶ໬ࡋࡓ⣽⬊ࡀⅣ⣲※ࡸ❅⣲※ ࡜࡞ࡿᰤ㣴ࡢ࡞࠸PBS ୰࡛ᅇ᚟ࡍࡿࡇ࡜ࡣ⪃࠼㞴࠸ࠋOhshima ࡽ(2013)ࡣࠊ㧗ᅽ ࡟ࡼࡾ୙άᛶ໬ࡋࡓ⣽⬊ࡀ PBS ୰࡛ᅇ᚟ࡍࡿ࣓࢝ࢽࢬ࣒ࢆゎᯒࡋࡓࠋᙼࡽࡣࠊ 㧗ᅽ࡟ࡼࡾぢ࠿ࡅୖ୙άᛶ໬ࡉࢀࡓ⣽⬊⩌࡟࠾࠸࡚ࠊ㧗ᅽ࡟ࡼࡿᦆയࢆཷࡅ࡚ ࠸ࡿࡀ୙άᛶ໬ࡋ࡚࠸࡞࠸⣽⬊ࡀഹ࠿࡟Ꮡᅾࡍࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋ㧗ᅽᦆ യ⣽⬊ࡣࠊ4°C ࡛ಖᏑࡋ࡚࠸ࡿሙྜ࡟࠾࠸࡚ࡣᅇ᚟ࡍࡿࡇ࡜ࡣ࡞࠸ࠋࡋ࠿ࡋࠊ 25°C ࡢ ᗘ᮲௳࡛ࡣࠊ㧗ᅽᦆയ࠿ࡽᅇ᚟ࡋࠊࡑࡢᚋࠊ࿘ᅖࡢ୙άᛶ໬ࡋࡓ⣽⬊ ࢆᰤ㣴※࡜ࡋ࡚㧗ᅽฎ⌮ࡍࡿ๓ࡢ⣽⬊ᩘࡢ 50%⛬ᗘࡲ࡛ቑṪࡍࡿࡇ࡜ࡀ᫂ࡽ࠿ ࡜࡞ࡗࡓࠋࡇࢀࡽࡢሗ࿌ࡣࠊ୙άᛶ໬ࡋࡓ⣽⬊࡛࠶ࡗ࡚ࡶ ᗘ᮲௳࡟ࡼࡗ࡚ࡣᅇ ᚟ࡋࠊቑṪࡍࡿࡇ࡜ࢆ♧ࡋ࡚࠾ࡾࠊ㣗ရຍᕤ࡟㧗ᅽฎ⌮ࢆᛂ⏝ࡍࡿሙྜࠊฎ⌮ᚋ ࡢ ᗘ⟶⌮ࡀ㔜せ࡛࠶ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋ

5 ➨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❧ࡉࡏࡿ࡜ࠊ࠾ࡼࡑ 76 cm ࡢ㧗ࡉࡲ࡛ࡀỈ㖟࡛ࡑࢀࡼࡾୖࡢ㒊ศࡀ┿✵࡜࡞ࡿࡇ࡜ࢆ᫂ࡽ࠿࡟ࡋࡓࠋ ࡇࡢཎ⌮ࢆᛂ⏝ࡋࡓỈ㖟ẼᅽィࢆⓎ᫂ࡋࡓຌ⦼࠿ࡽࠊᅽຊࡢ༢఩ࠕࢺࣝ; Torrࠖ ࡣTorriceli ࡢྡ࡟ࡕ࡞ࢇ࡛࠸ࡿࠋࡑࡋ࡚ࡑࡢ 4 ᖺᚋࡢ 1648 ᖺࠊPascal ࡟ࡼࡿ኱ Ẽᅽࡢㄆ㆑࡟ࡼࡗ࡚ᅽຊ࡜࠸࠺ᴫᛕࡀึࡵ࡚⛉Ꮫྐ࡟グࡉࢀࡓ(Pascal, 1653)ࠋ Pascal ࡣࠊỈ㖟Ẽᅽィࢆ⏝࠸࡚ࠊᆅ⾲࡜ᩍ఍ࡢᒇ᰿ࠊᆅ⾲࡜ࣆࣗ࢖࣭ࢻ࣭ࢻ࣮࣒ ᒣࡢ㡬ୖ࡟࠾࠸࡚Ỉ㖟ᰕࡢ㧗ࡉࡀኚ໬ࡍࡿࡇ࡜ࢆぢฟࡋࠊ኱ẼᅽࡢᏑᅾࢆド᫂ ࡋࡓࠋࡲࡓᙼࡣࠊᅽຊ(pressure)ࡀᅽ㏕(press)࡜␗࡞ࡿῶᑡ࡛࠶ࡿࡇ࡜ࢆ ͆⼚ࡣ ᣦ࡟ᣳࢇ࡛ࡕࡻࡗ࡜ᢲࡏࡤࡘࡪࢀࡿࡀ㧗࠸ᅽຊࡢୡ⏺࡛ࡣࡘࡪࢀ࡞࠸͇࡜ㄝ᫂ ࡋ࡚࠸ࡿ(ᯘ, 2008)ࠋ኱Ẽᅽࢆㄆ㆑ࡋࡓຌ⦼࠿ࡽࠊ௒᪥࡛ࡣᅽຊ༢఩ࡢࡇ࡜ࢆࠕࣃ ࢫ࢝ࣝ; Paࠖ࡜࿧ࡪࠋ 1905 ᖺ࡟ࡣࠊ㧗ᅽ◊✲ࡢṔྐ࡟ṧࡿ❅⣲࡜Ỉ⣲࠿ࡽ࢔ࣥࣔࢽ࢔ࡢ໬Ꮫྜᡂࡀ ᡂຌࡋࡓࠋHaber ࡣᖖ ᖖᅽ࡛ࡣ㐍⾜ࡋ࡞࠸ࡇࡢ໬Ꮫ཯ᛂ࡟ᑐࡋ࡚ࠊ200-500°Cࠊ 20-100 MPa ࡜࠸࠺㧗 㧗ᅽ᮲௳࡜㕲ࢆ୺య࡜ࡋࡓゐ፹ࢆ⏝࠸ࡿࡇ࡜࡟ࡼࡗ࡚࢔ ࣥࣔࢽ࢔ྜᡂἲࢆ☜❧ࡋࡓ(㔜ᯇ, 2013)ࠋࡑࡢᚋࠊBosch ࡟ࡼࡾࣁ࣮ࣂ࣮࣭࣎ࢵࢩ

6 ࣗἲ࡜࿧ࡤࢀࡿ࢔ࣥࣔࢽ࢔໬Ꮫྜᡂࡀၟᴗ໬ࡉࢀࡓࠋᮏἲࡣ௒᪥࡟࠾࠸࡚ࡶ᭱ ࡶຠ⋡ࡢࡼ࠸࢔ࣥࣔࢽ࢔ࡢ໬Ꮫྜᡂ᪉ἲ࡛࠶ࡾࠊᖺ㛫1 ൨ 8,700 ࢺࣥࡢ࢔ࣥࣔࢽ ࢔ែ❅⣲ࡀᮏἲ࡛⏕⏘ࡉࢀ࡚࠸ࡿ(Galloway et al., 2008)ࠋ⏕⏘ຠ⋡ࡢⰋ࠸ᑠ㯏ࡢ ᱂ᇵ࡟ࡣ❅⣲ศࢆྵࡴ኱㔞ࡢ⫧ᩱࡢ౪⤥ࡀ୙ྍḞ࡛࠶ࡿࡀࠊᙜ᫬ࡢ୺せ࡞❅⣲ ⫧ᩱࡣ༡⡿➼࡛᥇᥀ࡉࢀࡿ◪▼࡟㢗ࡗ࡚࠾ࡾࠊᑠ㯏ࡢ኱㔞⏕⏘ࡣ㞴ࡋ࠿ࡗࡓࠋࣁ ࣮ࣂ࣮࣭࣎ࢵࢩࣗἲ࡟ࡼࡾྜᡂࡉࢀࡓ࢔ࣥࣔࢽ࢔ࢆ⫧ᩱ࡜ࡋ࡚⏝࠸ࡿࡇ࡜࡛ࠊࡑ ࡢࡲࡲ࡛ࡣ⪔స࡟㐺ࡉ࡞࠸⑭ࡏࡓᅵᆅ࡟࠾࠸࡚ࡶᑠ㯏➼ࡢ✐≀ࡢ⏕⏘ࡀྍ⬟࡜ ࡞ࡾࠊୡ⏺ࡢᛴ⃭࡞ேཱྀቑຍ࡜♫఍ࡢⓎᒎ࡟㈉⊩ࡋࡓࠋࡇࡢຌ⦼࡟ࡼࡾࠊHaber ࡣ✵Ẽ࡜Ỉ࠿ࡽࣃࣥࢆసࡗࡓ⏨࡜ࡋ࡚ࡶ▱ࡽࢀ࡚࠾ࡾࠊ1918 ᖺ࡟ࣀ࣮࣋ࣝ໬Ꮫ ㈹ࢆཷ㈹ࡋ࡚࠸ࡿࠋ1931 ᖺ࡟ࡣ Bosch ࡶࠕ㧗ᅽ໬Ꮫ཯ᛂࡢ◊✲ࠖ࡟ࡼࡾࣀ࣮࣋ ࣝ໬Ꮫ㈹ࢆཷ㈹ࡋ࡚࠸ࡿࠋᮏἲࡣ㧗ᅽࢆ฼⏝ࡋࡓ໬Ꮫࣉࣟࢭࢫ࡜ࡋ࡚Ṕྐⓗ࡟ ᭱ࡶ᭷ྡ࡛࠶ࡾࠊ࠿ࡘே㢮࡟ࡶࡓࡽࡋࡓ㈉⊩ࡶ኱ࡁ࠸ࡶࡢ࡛࠶ࡿ(㔜ᯇ, 2013)ࠋ 19 ୡ⣖ᚋ༙࡟࡞ࡿ࡜ᚤ⏕≀࡬ࡢ㧗ᅽຠᯝ࡟ࡘ࠸࡚◊✲ࡀ㐍ࢇࡔࠋRegnard ࡣ ⣙300 MPa ࡢ㧗ᅽࢆⓎ⏕ࡉࡏࡿᅽຊ⿦⨨ࡢ㛤Ⓨ࡟ᡂຌࡋࡓࠋ1884 ᖺࠊᙼࡣࡇࡢ 㧗ᅽ⿦⨨ࢆ⏝࠸࡚ 6,000 m ࡢ῝ᾏ࡜ྠ⛬ᗘࡢᅽຊ᮲௳(⣙ 60 MPa)࡟࠾࠸࡚ୡ⏺ ࡛ึࡵ࡚ࣅ࣮ࣝ㓝ẕࡢᇵ㣴ࢆヨࡳࡓ(Regnard, 1884)ࠋࡑࡢ⤖ᯝࠊ㧗ᅽ᮲௳ୗ࡟࠾ ࠸࡚ࡶ㓝ẕࡢ࢚ࢱࣀ࣮ࣝⓎ㓝ࡀᘬࡁ㉳ࡇࡉࢀࡿࡇ࡜ࢆⓎぢࡋࡓࠋᙼࡢ◊✲ࡣ Buchner ࡟ࡼࡿ↓⣽⬊࡛ࡢⓎ㓝ࡢⓎぢ࡟㛫᥋ⓗ࡟ࡘ࡞ࡀࡿࠋBuchner ࡣࠊ1897 ᖺ

7 ࡟ 40-50 MPa ࡢᅽຊ᮲௳࡛㓝ẕࢆᅽᦢࡋ࡚ᚓࡓ㓝ẕᢳฟᾮࡀⅣỈ໬≀ࢆⓎ㓝ࡍ ࡿࡇ࡜ࢆሗ࿌ࡋࡓࠋᙼࡣࠊⓎ㓝ᕤ⛬࡟࠾࠸࡚⏕Ꮡࡋ࡚࠸ࡿᚤ⏕≀ࡀᚲࡎࡋࡶᚲせ ࡛ࡣ࡞ࡃࠊ㓝ẕࡢ⏕⏘ࡍࡿࢳ࣐࣮ࢮ(㓝⣲)ࡀⓎ㓝࡟㔜せ࡛࠶ࡿࡇ࡜ࢆⓎぢࡋࡓࠋ 1907 ᖺ࡟ࠕ↓⣽⬊࡛ࡢⓎ㓝ࡢⓎぢ࡜⏕໬Ꮫ࡟ࡼࡿ◊✲ࠖࡢᡂᯝ࡟ࡼࡾࠊࣀ࣮࣋ ࣝ໬Ꮫ㈹ࢆཷ㈹ࡋࡓࠋᙼࡣ㧗ᅽ᮲௳ୗ࡟࠾ࡅࡿ㓝⣲Ꮫࡢඛ㥑⪅࡜ࡋ࡚▱ࡽࢀ࡚ ࠸ࡿ(Jaenicke, 2007)ࠋ Roger ࡣࠊ1892 ᖺ࡟㧗ᅽ᮲௳࡟࠾ࡅࡿ⣽⳦ࡢ୙άᛶ໬࡟ࡘ࠸࡚ࡢ 2 ಶࡢ㔜せ ࡞ሗ࿌ࢆࡋࡓ(Roger, 1892; Roger, 1895)ࠋ1 ಶ┠ࡣࠊ㧗ᅽ᮲௳ୗࡢ୙άᛶ໬ᣲືࡀ ᚤ⏕≀ࡢᒓ✀࡟ࡼࡗ࡚␗࡞ࡿ࡜࠸࠺ሗ࿌࡛࠶ࡿࠋ౛࠼ࡤࠊStaphylococcus aureus ࡣ 300 MPa ⛬ᗘ࡛ࡣ኱ࡁ࡞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࠸ࡇ࡜ࢆሗ࿌ࡋࡓࠋࡇࢀࡽࡢሗ࿌ࡣࠊ㧗ᅽ࡟ࡼࡗ࡚ᚤ⏕≀ࡀ୙άᛶ໬ࡍࡿࡇ࡜ ࢆึࡵ࡚♧ࡋࡓሗ࿌࡛࠶ࡾࠊᙼࡢⓎぢ௨᮶ࠊᵝࠎ࡞ᚤ⏕≀✀ࡢᅽຊ⪏ᛶࡀ◊✲ࡉ ࢀࠊ㔜せ࡞▱ぢࡀ⵳✚ࡉࢀ࡚࠸ࡿࠋ

8 ➨4 㡯 㧗ᅽ◊✲ࡢṔྐ㸫㣗ရ࡬ࡢ㧗ᅽຍᕤࡢᛂ⏝㸫 ୡ⏺࡛ึࡵ࡚㣗ရ࡬ࡢ㧗ᅽฎ⌮ࢆሗ࿌ࡋࡓࡢࡣ Hite (1899 ᖺ)࡛࠶ࡿࠋᙼࡣࠊ ࣑ࣝࢡࡢ㛗ᮇಖᏑࡢࡓࡵ࡟⇕ẅ⳦௨እࡢ࢔ࣉ࣮ࣟࢳࢆ⪃᱌ࡋࡓ᭱ึࡢே≀࡛࠶ ࡿࠋ463 MPaࠊ1 h ࡢ᮲௳࡛㧗ᅽฎ⌮ࡉࢀࡓ࣑ࣝࢡࡣࠊᑡ࡞ࡃ࡜ࡶ 24 h ࡣ㓟ᛶ໬ ࡀᢚไࡉࢀ࡚⏑࿡ࡀಖᣢࡉࢀࡿࡇ࡜ࢆⓎ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Ⓨ ࡋࡓࠋࡑࢀ࡟ࡼࡾ 1912 ᖺ࡟㧗ᅽୗ࡟࠾ࡅࡿỈࡢ≧ែኚ໬ࡢ┦㛵ᅗࢆሗ࿌ࡋࡓ (Bridgman, 1912)ࠋ1914 ᖺ࡟ࡣࠊ㭜༸࡟ 500~700 MPaࠊ30~60 min ࡢ᮲௳࡛㧗ᅽฎ ⌮ࡍࡿࡇ࡜࡟ࡼࡾࠊ༸Ẇࢆ๭ࡿࡇ࡜࡞ࡃࠊ༸㯤࣭༸ⓑࡀจᅛࡍࡿࡇ࡜ࢆⓎぢࡋࡓ (Bridgman, 1914)ࠋᙼࡢሗ࿌ࡣ㧗 ࡛ほᐹࡉࢀࡿࢱࣥࣃࢡ㉁ࡢኚᛶࡀ㠀⇕ⓗ᮲௳ ࡢ㧗ᅽ࡛ࡶྠᵝ࡟ᘬࡁ㉳ࡇࡉࢀࡿࡇ࡜ࢆึࡵ࡚᫂ࡽ࠿࡜ࡋࡓ⏬ᮇⓗ࡞Ⓨぢ࡛࠶ ࡾࠊࡑࡢᚋࡢࢱࣥࣃࢡ㉁ࡢ㧗ᅽኚᛶ࣓࢝ࢽࢬ࣒࡟㛵ࡍࡿ◊✲ࡢඛ㥑ࡅ࡜࡞ࡗࡓࠋ Bridgman ࡣࠊࠕ㉸㧗ᅽ⿦⨨ࡢ㛤Ⓨ࡜ࡑࢀ࡟ࡼࡿ㧗ᅽ≀⌮Ꮫ࡟㛵ࡍࡿⓎぢࠖ࡟ࡼࡾ 1946 ᖺ࡟ࣀ࣮࣋ࣝ≀⌮Ꮫ㈹ࢆཷ㈹ࡋࠊ㧗ᅽ≀⌮Ꮫࡢ∗࡜࿧ࡤࢀ࡚࠸ࡿࠋ ࡋ࠿ࡋࠊHite 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ࡳࡽࢀࡿࡇ࡜ࡣ

9

࡞࠿ࡗࡓ(㕥ᮌ, 2013)ࠋ

㣗ရ࡬ࡢ㧗ᅽ฼⏝ࡢ㌿ᶵ࡜࡞ࡗࡓࡇ࡜ࡀ 1968 ᖺ࡟㉳ࡇࡗࡓ◊✲⏝₯ỈⰄ Alvin ྕࡢỿἐ஦௳࡛࠶ࡿ(Pope, 1973)ࠋAlvin ྕࡣ Woods Hole Oceanographic Institution (WHOI)࡟ᡤᒓࡍࡿ₯ỈⰄ࡛࠶ࡿࠋ1968 ᖺࡢ෤ࠊAlvin ྕࡣᅇ⯟㏵୰࡟ ஦ᨾ࡟㐼㐝ࡋࡓࠋᖾ࠸࡟ࡶ஌ဨࡣ඲ဨ⬺ฟ࡛ࡁࡓࡀ⯪యࡣ1,543 m ࡢ῝ᾏ࡬ỿࢇ ࡔࠋAlvin ྕࡀ῝ᾏ࠿ࡽᘬࡁᥭࡆࡽࢀࡓࡢࡣࠊࡑࡢ஦ᨾ࠿ࡽ 10 ࠿᭶ᚋ࡛࠶ࡿࠋࡑ ࡢᚋࡢ⯪యࡢㄪᰝ࡟࠾࠸࡚ࠊ㦫ࡃ࡭ࡁࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ⯪య࡜ඹ࡟῝ᾏ࡬ ỿࢇ࡛࠸ࡓࣜࣥࢦࡸࢧࣥࢻ࢘࢕ࢵࢳ➼ࡢእぢࠊ࿡ࠊ㤶ࡾࡀỿἐࡍࡿ๓࡜࡯࡜ࢇ࡝ ኚ໬ࡋ࡚࠸࡞࠿ࡗࡓࡢ࡛࠶ࡿࠋࡑࢀࡔࡅ࡛ࡣ࡞ࡃࠊᚤ⏕≀Ꮫⓗ࣭⏕໬Ꮫⓗ࡟ࡶ㣗 ရࡢရ㉁ࡀⰋࡃಖᣢࡉࢀ࡚࠸ࡓࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓࠋ୍⯡ⓗ࡟ࡣࠊ4°C ⛬ᗘࡢ ෭ⶶ᮲௳࡟࠾࠸࡚ࡣࠊࢹࣥࣉࣥࡸࢱࣥࣃࢡ㉁➼ࡣᩘ㐌㛫࡛ຎ໬ࡋ࡚⭉ᩋࡍࡿࠋ Alvin ྕࡢ⯪య࡛ಖᏑࡉࢀ࡚࠸ࡓ㣗ရࡢရ㉁ࡣࠊ῝ᾏ≉᭷ࡢప ࠊ㈋ᰤ㣴᮲௳ࡢ ௚࡟ᆅ⾲ࡢ኱Ẽᅽ(⣙ 0.1 MPa)࡜ẚ㍑ࡋ࡚ 100 ಸ௨ୖ㧗࠸ 15 MPa ⛬ᗘࡢᅽຊ࡟ ࡼࡗ࡚⥔ᣢࡉࢀ࡚࠸ࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡓࠋࡇࡢ▱ぢࡣࠊ㧗ᅽ᮲௳ࡀ⭉ᩋࢆᘬࡁ ㉳ࡇࡍᚤ⏕≀ࡢ௦ㅰάᛶࢆᢚไࡍࡿ࡟ࡶ㛵ࢃࡽࡎࠊ㣗ရࡢရ㉁ࢆຎ໬ࡉࡏ࡞࠸ ࡇ࡜ࢆ♧ࡋ࡚࠾ࡾࠊ㣗ရࡢప ẅ⳦࡬ࡢ㧗ᅽࡀ฼⏝࡛ࡁࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋ Alvin ྕࡢỿἐ஦ᨾ௨᮶ࠊ㧗ᅽᢏ⾡ࡢ㣗ရຍᕤ࡬ࡢᛂ⏝ࡢྍ⬟ᛶࡀ◊✲ࡉࢀࠊ 1987 ᖺ࡟ᯘ ຊ୸࡟ࡼࡾ㣗ရຍᕤ࡬ࡢ㧗ᅽ฼⏝ࡀᥦၐࡉࢀࡓ(Hayashi et al., 1987)ࠋ

10 㧗ᅽࡣࠊ⏕యศᏊࡢ㠀ඹ᭷⤖ྜࡢࡳ࡟స⏝ࡍࡿࡓࡵ࡟ࠊձ㣗ရ⣲ᮦࡀᣢࡘ᪂㩭࡞ 㢼࿡ࠊⰍࠊ࿡ࢃ࠸ࢆಖᣢ࡛ࡁࡿࠊղຍ⇕࡟ࡼࡾ◚ቯࡉࢀࡿࣅࢱ࣑ࣥ C ➼ࡢ᭷⏝ ᡂศࡢຎ໬ࡀᑡ࡞࠸ࠊճຍ⇕࡟ࡼࡿ࢔ࢡࣜࣝ࢔࣑ࢻ➼ࡢ␗ᖖ≀㉁ࡸ␗⮯ࡀⓎ⏕ ࡋ࡞࠸ࠊմຍ⇕ຍᕤ࡜ࡣ␗࡞ࡿ⊂≉࡞≀ᛶࡀ⏕ࡌࡿࠊյຍ⇕ฎ⌮࡟ẚ࡭࡚┬࢚ࢿ ࣝࢠ࣮࡛࠶ࡿ➼ࡢ฼Ⅼࡀ࠶ࡿ(ᯘ, 1991)ࠋࠕ㣗ရຍᕤ࡬ࡢ㧗ᅽ฼⏝ࠖࡢᥦၐ௨㝆ࠊ ከࡃࡢ◊✲⪅࡟ࡼࡗ࡚㧗ᅽᢏ⾡ࢆᛂ⏝ࡋࡓప ẅ⳦ᢏ⾡ࠊ㣗ရຍᕤ࣭〇㐀ᢏ⾡ࠊ 㓝⣲άᛶࡸࡑࢀ࡟ࡼࡿ᭷⏝ᡂศࡢቑᙉ➼ࡢ◊✲ࡀ᥎㐍ࡉࢀࡓࠋ ᪥ᮏᅜෆ࡛ࡣ㎰ᯘỈ⏘┬࡟ࡼࡾࠕ㣗ရ⏘ᴗ㉸㧗ᅽ฼⏝ᢏ⾡◊✲⤌ྜࠖࡀ⤌⧊ࡉ ࢀࠊࠕ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┴ࡢ1 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⏕ࢥࣥࢯ࣮ࢩ࢔࣒㸫㧗ᅽฎ ⌮ࢆ฼⏝ࡋࡓ᪂つᶵ⬟ᛶ㣗ᮦࡢ㛤Ⓨ࡜⏘ᴗ໬ࠖࡀ᥇ᢥࡉࢀࠊ2007 ᖺ࡟ࡣ㧗ᅽᢏ ⾡࡟㛵ࡍࡿᡓ␎ⓗ࡞ᶆ‽໬ࢆ᥎㐍ࡍࡿࡓࡵࡢࠕ㧗ᅽᇶ┙ᢏ⾡ᶆ‽໬ᶵᵓࠖࢆ❧ࡕ

11 ୖࡆ࡚࠸ࡿࠋ2008 ᖺ࡟ࡣࠊ᪥ᮏ⛉Ꮫᢏ⾡᣺⯆ᶵᵓ (JST) 2007 ᖺᗘࠕ᪂₲┴ᆅᇦ ⤖㞟ᆺ◊✲ࣉࣟࢢ࣒ࣛ㸫㣗ࡢ㧗௜ຍ౯್໬࡟㈨ࡍࡿᇶ┙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)࡟ࡼࡗ࡚ࡲ࡜ࡵࡽࢀ࡚࠸ࡿࠋ 㧗ᅽ◊✲ࡢᡂᯝࢆⓎ⾲ࡋ࡚㆟ㄽࡋྜ࠺ሙ࡜ࡋ࡚ࠊ⏕≀㛵㐃㧗ᅽ◊✲఍ࡀ 1988 ᖺ࡟タ❧ࡉࢀࡓࠋྠᖺ࡟➨1 ᅇࢩ࣏ࣥࢪ࣒࢘ࡀி㒔࡛ࠊ2013 ᖺ࡟ࡣ➨ 18 ᅇࢩࣥ ࣏ࢪ࣒࢘ࢆᒱ㜧኱Ꮫ࡛㛤ദࡍࡿࡇ࡜ࡀ࡛ࡁࡓࠋ2015 ᖺ࡟ࡣᗈᓥ࡛➨ 20 ᅇࢩ࣏ࣥ ࢪ࣒࢘ࡀ㛤ദࡉࢀࡿணᐃ࡛࠶ࡿࠋᅜእ࡟┠ࢆྥࡅࡿ࡜International Conference on High Pressure Bioscience and Biotechnology (HPBB)ࡀ 2000 ᖺ࡟タ❧ࡉࢀࠊ௨㝆 2 ᖺẖ࡟ୡ⏺ྛᆅ࡛㛤ദࡉࢀ࡚࠸ࡿ(Nomura and Iwahashi, 2014)ࠋ2014 ᖺ࡟➨ 8 ᅇ ኱఍ࡀࣇࣛࣥࢫ࡛㛤ദࡉࢀࡓࠋ2016 ᖺ࡟ࡣ➨ 9 ᅇ኱఍ࡀ࢝ࢼࢲ࡛㛤ദࡉࢀࡿண ᐃ࡛࠶ࡿࠋ

12 ➨ ➨2 ⠇ 㓝ẕ 㓝ẕ(Yeast)ࡣࠊ┤ᚄ 5~10 ȣm ⛬ᗘࡢ༸ᆺ࡛ࠊฟⱆࡸศ⿣࡟ࡼࡗ࡚ቑṪࡍࡿ┿ ᰾༢⣽⬊⏕≀࡛࠶ࡿࠋ㓝ẕ࡟ࡣࣄࢺ࡜ྠࡌࡼ࠺࡟ᑑ࿨ࡶᏑᅾࡍࡿࠋ㟁Ꮚ㢧ᚤ㙾࡛ ฟⱆ㓝ẕࢆほᐹࡍࡿ࡜ࠊ⣽⬊ࡢ⾲㠃࡟ፉ㓝ẕࡀฟⱆࡍࡿ᫬࡟⏕ࡌࡓฟⱆ⑞ࠊ⮬㌟ ࡀฟⱆࡋࡓ᫬࡟⏕ࡌࡓฟ⏕⑞࡜࿧ࡤࢀࡿ」ᩘࡢพฝࡀ☜ㄆ࡛ࡁࡿ(㔝ᮧ, ᒾᶫ, 2013)ࠋࡇࡢฟⱆ⑞ࡀ⣽⬊ࡢ⾲㠃࡟ṧࡿࡓࡵࠊࡑࡢᩘࡀฟⱆᅇᩘࠊ༶ࡕ㓝ẕࡢᑑ ࿨࡟┦ᙜࡍࡿࠋ⣙20 ᅇฟⱆࡍࡿ࡜ࠊࡑࢀ௨ୖࡣฟⱆࡍࡿࡇ࡜ࡀ࡛ࡁ࡞ࡃ࡞ࡿࡇ ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Egilmez and Jazwinski, 1989)ࠋฟⱆᅇᩘࡣ⏕⌮ⓗ࡞せᅉ࡛ᙳ 㡪ࢆཷࡅࡿࡇ࡜ࡶ࠶ࡾࠊ⎔ቃࢫࢺࣞࢫ➼࡛ᐜ᫆࡟ኚ໬ࡍࡿࠋࡲࡓࠊ㓝ẕࡣ〇ࣃࣥ

ࡸ㔊㐀➼ࡢ㣗ရ⏘ᴗศ㔝࡟ᗈࡃ⏝࠸ࡽࢀ࡚࠸ࡿࡔࡅ࡛࡞ࡃࠊࣂ࢖࢜ࢸࢡࣀࣟࢪ

࣮ศ㔝࡟࠾࠸࡚ࡶࣂ࢖࢚࢜ࢱࣀ࣮ࣝࡢ⏕⏘➼࡟㔜せ࡞ᙺ๭ࢆᯝࡓࡋ࡚࠸ࡿ

13 ➨1 㡯 ࣔࢹࣝ⏕≀࡜ࡋ࡚ࡢ㓝ẕ ᭱ࡶ୍⯡ⓗ࡞㓝ẕ࡛࠶ࡿ Saccharomyces cerevisiae ࡣࠊࣄࢺ࡜ྠࡌ┿᰾⏕≀࡛ ࠶ࡿୖ࡟ࢤࣀ࣒ᵓ㐀ࡀẚ㍑ⓗ༢⣧࡛࠶ࡿࡢ࡛ࠊ኱⭠⳦➼ࡢཎ᰾⏕≀࡛ᚓࡓ▱ぢ ࡜ẚ㍑ࡋ࡚ࠊ㓝ẕࡢศᏊ⏕≀Ꮫⓗ▱ぢࢆࣄࢺ࡬ᛂ⏝ࡋ᫆࠸࡜࠸࠺฼Ⅼࡀ࠶ࡿࠋࡲ ࡓࠊୡ௦᫬㛫ࡀ▷ࡃࠊᏳ౯࡞ᇵᆅ࡛ቑṪ࡛ࡁࠊẘᛶࡶ↓ࡃࠊ⏕⌮≧ែࡢ㧗࠸෌⌧ ᛶࡀᮇᚅ࡛ࡁࡿࡓࡵ࡟኱Ꮫ➼ࡢᐇ㦂ᐊࡢタഛ࡛ẚ㍑ⓗ⡆༢࡟◊✲࡛ࡁࡿࠋࡇࢀ ࡽࡢ฼Ⅼ࠿ࡽ㓝ẕࡣศᏊ⏕≀Ꮫࢆጞࡵᵝࠎ࡞ศ㔝ࡢࣂ࢖࢜࢔ࢵࢭ࢖ࡢࣔࢹࣝ⏕

≀࡜ࡋ࡚฼⏝ࡉࢀ࡚࠸ࡿ(Haney et al., 2001; Matsuoka et al., 2005; Iwahashi et al., 2007; Yasokawa and Iwahahsi, 2010)ࠋ

1996 ᖺ࡟ࡣࠊ┿᰾⏕≀࡜ࡋ࡚ึࡵ࡚ S. cerevisiae ࡢ඲ࢤࣀ࣒ࡢሷᇶ㓄ิ᝟ሗࡀ ゎㄞࡉࢀࡓ(Goffeau et al.,1996)ࠋ㓝ẕࡢ⣙ 6000 ✀㢮ࡢ㑇ఏᏊ࡟ࢥ࣮ࢻࡉࢀ࡚࠸ ࡿࢱࣥࣃࢡ㉁ࡢ࠺ࡕࠊ࠾ࡼࡑ 80%ࡀ⏕Ꮡ࡟ᚲ㡲࡛࡞࠸ࡇ࡜ࡶ᫂ࡽ࠿࡜࡞ࡗ࡚࠸ ࡿࠋࡲࡓࠊ㓝ẕࢤࣀ࣒࡟ࡼࡗ࡚ࢥ࣮ࢻࡉࢀ࡚࠸ࡿࢱࣥࣃࢡ㉁ࡢᑡ࡞ࡃ࡜ࡶ 31% ࡣࣄࢺ࡜࣍ࣔࣟࢢࢆᣢࡗ࡚࠾ࡾࠊ㏫࡟㑇ఏ⑓࡟㛵㐃ࡍࡿࣄࢺࢤࣀ࣒ࡢ⣙ 50%ࡢ 㑇ఏᏊࢆࠊ㓝ẕࡣ࣍ࣔࣟࢢ࡜ࡋ࡚ᣢࡗ࡚࠸ࡿࡇ࡜ࡶሗ࿌ࡉࢀ࡚࠸ࡿ(Hartwell, 2004)ࠋࡍ࡞ࢃࡕࠊ㓝ẕ࡜ࣄࢺࡢࢱࣥࣃࢡ㉁ࡢከࡃࡣྠᵝ࡟ᶵ⬟ࡋ࡚࠾ࡾࠊࢫࢺ ࣞࢫᛂ⟅࣓࢝ࢽࢬ࣒ゎ᫂ࡢࡓࡵࡢࣔࢹࣝ⏕≀࡜ࡋ࡚ࡶ◊✲࡟฼⏝ࡉࢀ࡚࠸ࡿ

14 ࡓࠊ㓝ẕࡢ඲ࢤࣀ࣒ゎᯒᚋࠊ඲㑇ఏᏊࢆᦚ㍕ࡋࡓDNA ࢳࢵࣉࡀ᪩ᛴ࡟౪⤥ࡉࢀ ࡓ(ᒾᶫ, 2002)ࠋࡑࢀᨾ࡟ࢤࣀ࣑ࢡࢫゎᯒ⤖ᯝࡢ⵳✚ࡀ㇏ᐩ࡟࠶ࡾࠊゎᯒࢆᐜ᫆ ࡟ࡋ࡚࠸ࡿࠋ㓝ẕࢤࣀ࣑ࢡࢫࡢ୍␒኱ࡁ࡞฼Ⅼࡣࠊྛ㑇ఏᏊࡢᶵ⬟᝟ሗࡢ㇏ᐩࡉ ࡛࠶ࡾࠊࡉࡽ࡟ゎᯒࢆᐜ᫆࡟ࡋ࡚࠸ࡿࠋ 㓝ẕ࡜ࣄࢺࡢ࿧྾ᶵ⬟࡟㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡣẚ㍑ⓗࡼ ࡃಖᏑࡉࢀ࡚࠾ࡾࠊ࢚ࢿࣝࢠ࣮⏕⏘ࡢࡓࡵࡢ㓟໬ⓗࣜࣥ㓟໬ࡶྠᵝࡢᶵ⬟ࢆ᭷ ࡍࡿࠋຍ࠼࡚ࠊ㓝ẕࡣ࢚ࢱࣀ࣮ࣝⓎ㓝࡟ࡼࡾቑṪࡍࡿࡇ࡜ࡀ࡛ࡁࡿࡢ࡛ࠊ࣑ࢺࢥ ࣥࢻࣜ࢔ࡀኚ␗ࡋ࡚࿧྾ᶵ⬟ࡀḞኻࡋ࡚ࡶ⏕Ꮡࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿࠋࡑࡢࡓ ࡵ࡟࣑ࢺࢥࣥࢻࣜ࢔ࡀ㛵㐃ࡍࡿ㑇ఏ⑓ࡸ⒴ࢆጞࡵ࡜ࡋࡓከࡃࡢ⑓Ẽࡸ࿧྾㜼ᐖ ๣➼࡟ᑐࡍࡿᇶ♏◊✲ࡢࣔࢹࣝ⏕≀࡟ࡶ࡞ࡗ࡚࠸ࡿ (⚟ཎ, 1986)ࠋ ㏆ᖺࠊ㓝ẕࡀ⎔ቃ୰࡟ᬑ㐢ⓗ࡟Ꮡᅾࡍࡿ⏕≀ࡢ 1 ࡘ࡛࠶ࡿࡇ࡜ࡀ᫂ࡽ࠿࡟ࡉ ࢀࠊ㓝ẕࡢࢺࢥࢩࢥࢤࣀ࣑ࢡࢫࡀ⏕ែẘᛶホ౯࡟ᛂ⏝࡛ࡁࡿࡇ࡜ࡀሗ࿌ࡉࢀࡓ (Liti et al., 2009)ࠋࡇࢀ࡟ࡼࡾࠊ⎔ቃ୰ࡢṧ␃㎰⸆ࡸ㔠ᒓࢼࣀ⢏Ꮚ➼ࡢ⏕య࡬ࡢẘ ᛶホ౯࡟㓝ẕࢆ฼⏝ࡍࡿࡇ࡜ࡀྍ⬟࡜࡞ࡗࡓࠋ

15

➨2 㡯 Ⓨ㓝㣗ရ࡜ࡋ࡚ࡢ㓝ẕ

ᡃࠎே㢮ࡣࠊᚤ⏕≀࡜Ⓨ㓝࡜࠸࠺ᴫᛕࢆ⌮ゎࡋ࡚࠸࡞࠸ྂ௦࠿ࡽఏ⤫ⓗ࡟Ⓨ

㓝㣗ရࢆ⏕⏘ࡋ࡚ࡁࡓࠋ࢔ࢪ࢔࡛ࡣࠊᑠ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ရࡣ୺࡟ங㓟⳦࡟ࡼࡗ࡚⏕⏘ࡉࢀࡿࠋᮏ◊✲࡛⏝࠸ࡿS. cerevisiae ࡣࠊ࢔ࣝࢥ

࣮ࣝⓎ㓝࡟ࡼࡾ࣡࢖ࣥࠊࣅ࣮ࣝࠊΎ㓇➼ࡢ㓇㢮ࠊ࿡ჯࠊ㓺Ἔ➼ࡢㄪ࿡ᩱࠊࣃࣥࠊ

ₕ ≀ ➼ ࡢ ᵝ ࠎ ࡞ Ⓨ 㓝 㣗 ရ ࢆ ⏕ ⏘ ࡍ ࡿ ᭷ ⏝ ᚤ ⏕ ≀ ࡜ ࡋ ࡚ ᗈ ࡃ ▱ ࡽ ࢀ ࡚ ࠸ ࡿ

(Nomura and Iwahashi, 2014)ࠋࡇࢀࡽࡢఏ⤫ⓗ࡞Ⓨ㓝㣗ရࡣ୺㣗࡜ࡋ࡚ࡔࡅ࡛ࡣ ࡞ࡃࠊᢠ㓟໬άᛶࡸᢠ࢔ࣞࣝࢠ࣮ࠊච␿㈿ά➼ࡢ᭷┈࡞ຠᯝࡀᮇᚅ࡛ࡁࡿ೺ᗣ㣗

ရࡸࢥ࣑ࣗࢽࢣ࣮ࢩࣙࣥࢆ෇⁥࡟ࡍࡿႴዲရ࡜ࡋ࡚ࡶ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㉁ࢆຎ໬ࡉࡏ࡚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

16 ➨ ➨3 ⠇ 㧗ᅽ㣗ရຍᕤᢏ⾡  㧗ᅽ㣗ရຍᕤᢏ⾡࡜ࡣࠊᩘⓒ MPa ࡢ㧗ᅽ᮲௳࡟ࡼࡾ㣗ရࡢẅ⳦ࡸᶵ⬟ᛶᡂศ ࡢᐩ໬ࠊ㓝⣲άᛶࡢྥୖࠊ௜ຍ౯್ࡢ௜୚➼ࢆ┠ⓗ࡟㠀⇕ⓗ࠶ࡿ࠸ࡣຍ⇕࡜⤌ࡳ ྜࢃࡏࡿࡇ࡜࡟ࡼࡾࠊ㣗ရࢆຍᕤࡍࡿᢏ⾡ࡢ⥲⛠࡛࠶ࡿࠋ1987 ᖺ࡟ி㒔኱Ꮫᯘ ຊ୸ ຓᩍᤵ(ᙜ᫬)࡟ࡼࡾ㧗ᅽᢏ⾡ࢆ㣗ရຍᕤ࡟ᛂ⏝ࡍࡿࡇ࡜ࢆᥦၐࡉࢀࠊᮏᢏ 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ရࡢຍᕤ࡟ᛂ⏝࡛ࡁࡿ࡜࠸࠺฼Ⅼࡀ࠶ࡿࠋࡑࡢࡓࡵ࡟㧗ᅽ㣗 ရຍᕤࡣࠊ㠀⇕ⓗ࡟࠶ࡿ࠸ࡣᚑ᮶ࡢຍ⇕ຍᕤࡼࡾࡶప࠸ ᗘ࡛㣗ရࢆຍᕤࡋࠊᚤ ⏕≀ࢆ୙άᛶ໬ࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿࠋ㧗ᅽ㣗ရຍᕤࡣࠊࡇࢀࡲ࡛࡟ຍ⇕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 ᅽ࡟ࡼࡿ∻⾃ࡢẆࡴࡁ(Murokoshi, 2004)➼࡟ᛂ⏝ࡉࢀ࡚࠸ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ㧗 ᅽᢏ⾡࡟ࡼࡿᵝࠎ࡞㣗ရຍᕤࡸᚤ⏕≀ࡢ୙άᛶ໬࡟ࡣࠊ300 MPa ௨ୖࡢ㧗ᅽࡀ

17

ᚲせ࡛࠶ࡿࠋࡑࡢࡓࡵ࡟ࡣ㧗ᅽ࡟⪏࠼ࡿࡇ࡜ࡀ࡛ࡁࡿ㧗ࢥࢫࢺ࡞⪏ᅽᐜჾࢆ᭷

ࡍࡿ㧗ᅽ⿦⨨ࡀᚲ㡲࡛࠶ࡾࠊ㣗ရ࡬ࡢ㧗ᅽຍᕤࡣ㧗௜ຍ౯್ࢆ᭷ࡍࡿ㣗ရ࡟㝈

ࡽࢀ࡚࠾ࡾࠊ㧗ᅽᢏ⾡ࡢᬑཬࡀጉࡆࡽࢀ࡚࠸ࡿࡢࡀ⌧≧࡛࠶ࡿࠋ

➨2 㡯 Pressure Regulated Fermentation

 ㏆ᖺࠊ㣗ရ࡬ࡢ㧗ᅽຍᕤࢆࡼࡾᬑཬࡉࡏࡿࡓࡵ࡟ࠊࡼࡾప࠸ᅽຊ࡟ࡼࡾ㣗ရࡢ

Ⓨ㓝ࢆไᚚࡍࡿᢏ⾡(Pressure Regulated Fermentation; PReF)ࡢ㛤Ⓨࡀᥦ᱌ࡉࢀ࡚ ࠸ࡿ(Nomura and Iwahashi, 2014)ࠋⓎ㓝㣗ရࢆ⏕⏘ࡍࡿ㓝ẕࡢⓎ㓝ࡀไᚚ࡛ࡁ࡞ ࠸ሙྜࠊㄪ࿡ᾮࡢⓑ⃮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 ࢆᢚไࡍࡿࡓࡵ࡟ࠊྂࡃ࠿ࡽ㓝ẕ࡜ඹ࡟Ⓨ㓝㣗ရࡢ⏕⏘࡟฼⏝ࡉࢀ࡚ࡁࡓࠋPReF ᢏ⾡ࡣࠊ㧗ᅽᢏ⾡ࡢ฼Ⅼࢆά࠿ࡋࠊⓎ㓝㣗ရ୰࡟Ꮡᅾࡍࡿࣅࢱ࣑ࣥ C ➼ࡢ᭷⏝ ᡂศࢆ⥔ᣢࡋࡘࡘࠊᚤ⏕≀ࡢⓎ㓝ࢆไᚚࡍࡿࡇ࡜ࢆ┠ᣦࡋ࡚࠸ࡿࠋࡲࡓࠊPReF ᢏ⾡ࢆᛂ⏝ࡍࡿࡇ࡜ࡣࠊຍ⇕࡟ࡼࡿ᭷⏝≀㉁ࡢຎ໬ࡔࡅ࡛ࡣ࡞ࡃࠊሷࡸ㓟ࡢ㐣๫ ῧຍ࡟ࡼࡿ೺ᗣ⿕ᐖࡢⓎ⏕ࡶᢚไࡍࡿࡇ࡜ࡀᮇᚅ࡛ࡁࡿࠋేࡏ࡚ࠊPReF ᢏ⾡࡛ ࡣࠊ㧗ᅽ⿦⨨ࡢࢥࢫࢺࢆ๐ῶࡍࡿࡓࡵ࡟ࠊ100~200 MPa ⛬ᗘࡢ✜ࡸ࠿࡞୰㧗ᅽ㡿 ᇦࢆ⏝࠸ࡿࠋPReF ᢏ⾡ࡣࠊᩘⓒ MPa ௨ୖࡢ㧗ᅽࡀᚲせ࡞஺㞧ᚤ⏕≀ࡢ⁛⳦

18 (sterilization)ࢆࢱ࣮ࢤࢵࢺࡍࡿࡢ࡛ࡣ࡞ࡃࠊ୺せ࡞Ⓨ㓝ᚤ⏕≀࡛࠶ࡿ㓝ẕࡢẅ⳦ (pasteurization)࣭୙άᛶ໬ࢆࡍࡿࡇ࡜࡟ࡼࡾࠊ㐣Ⓨ㓝ࢆไᚚࡍࡿࡇ࡜ࢆ┠ⓗ࡜ࡍ ࡿ࢔ࣉ࣮ࣟࢳ࡛࠶ࡿࠋங㓟⳦ࡣ⮬㌟ࡢ⏕⏘ࡍࡿங㓟࡟ࡼࡿpH ࡢపୗ࡟ࡼࡾ୙ά ᛶ໬ࡍࡿࡓࡵࠊPReF ᢏ⾡࡟࠾࠸࡚ࡣࢱ࣮ࢤࢵࢺ࡜ࡋ࡞࠸ࠋࡇࢀࡽࡢࡇ࡜࠿ࡽ PReF ᢏ⾡ࡢ☜❧ࡓࡵ࡟ࡣࠊ୰㧗ᅽ᮲௳࡛୙άᛶ໬ࡍࡿᅽຊ࡟ᙅ࠸㓝ẕᰴࡢసฟ ࡀᚲせ࡛࠶ࡿࠋ ➨3 㡯 ᅽຊឤཷᛶ㓝ẕࡢసฟ ࡇࢀࡲ࡛࡟ᐇ㦂ᐊࣔࢹࣝ㓝ẕᰴS. cerevisiae KA31a ᰴࢆぶᰴ࡜ࡋ࡚ࠊࡑࢀࡼࡾ ࡶ㧗࠸ᅽຊឤཷᛶ⬟ࢆ♧ࡍᅽຊឤཷᛶኚ␗ᰴa924E1 ᰴࢆ⣸እ⥺↷ᑕἲ࡟ࡼࡿࣛ ࣥࢲ࣒✺↛ኚ␗ᑟධ࡟ࡼࡾྲྀᚓࡋࡓ(Shigematsu et al., 2010a)ࠋྲྀᚓࡋࡓ a924E1 ᰴࡣࠊKA31a ᰴ࡜ྠ➼ࡢ࢚ࢱࣀ࣮ࣝⓎ㓝⬟ࢆ᭷ࡋ࡚࠾ࡾࠊᅽຊࡔࡅ࡛࡞ࡃ ᗘ ࡟ᑐࡋ࡚ࡶឤཷᛶࢆ♧ࡍࡇ࡜ࡀሗ࿌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⏝࡟᭷┈࡛࠶ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ a924E1 ᰴࡣࣛࣥࢲ࣒✺↛ኚ␗࡟ࡼࡾྲྀᚓࡉࢀࡓࡓࡵࠊᅽຊឤཷᛶኚ␗ࡀᑟධࡉ ࢀࡓ㑇ఏᏊᗙ࠾ࡼࡧᅽຊឤཷᛶࡢᇶ♏࡜࡞ࡿ࣓࢝ࢽࢬ࣒ࡣࠊᮍࡔ᫂ࡽ࠿࡜࡞ࡗ ࡚࠸࡞࠸ࠋ

19 ➨ ➨4 ⠇ ◊✲┠ⓗ ᮏ◊✲ࡢ┠ⓗࡣࠊ㧗ᅽ⿦⨨ࡢタഛᢞ㈨ࢥࢫࢺࢆపῶࡍࡿࡓࡵ࡟ࠊ100㸫200 MPa ⛬ᗘࡢ୰㧗ᅽ࡟ࡼࡾ㓝ẕS. cerevisiae ࢆ୙άᛶ໬ࡍࡿ PReF ᢏ⾡ࢆసฟࡍࡿࡇ࡜ ࡛࠶ࡿࠋPReF ᢏ⾡࡟ࡼࡾⓎ㓝ᚤ⏕≀ࢆẅ⳦ࡋࡓᚋࡢⓎ㓝㣗ရࡣప ᮲௳࡛ಖᏑ࣭ ὶ㏻ࡉࢀࡿࠋᚑࡗ࡚ࠊPReF ᢏ⾡ࡢᐇ⏝໬࡟ࡣࠊ༑ศ࡞࢚ࢱࣀ࣮ࣝ⏕⏘⬟ࢆ᭷ࡋࠊ ᑦୟࡘ㧗ᅽ࡟ឤཷᛶࢆ♧ࡍ⏘ᴗ㓝ẕᰴࡢసฟࡀᚲ㡲࡜࡞ࡿࠋ ࡇࢀࡲ࡛࡟సฟࡋࡓᅽຊឤཷᛶኚ␗ᰴa924E1 ᰴࡣࠊᮍࡔ᫂ࡽ࠿࡜࡞ࡗ࡚࠸࡞ ࠸ᮍ▱ࡢせᅉ࡟ࡼࡾᅽຊឤཷᛶ⬟ࡀ௜୚ࡉࢀࡓྍ⬟ᛶࡀ࠶ࡿࠋᮏ◊✲࡛ࡣࠊᅽຊ ឤཷᛶࡢ⾲⌧ᆺࢆᘬࡁ㉳ࡇࡍኚ␗㑇ఏᏊ࠾ࡼࡧࡑࡢ࣓࢝ࢽࢬ࣒ࢆ DNA ࣐࢖ࢡ ࣟ࢔ࣞ࢖ゎᯒ࠾ࡼࡧ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒ࡟ࡼࡾゎᯒࡋࡓࠋ

20

➨

➨

2 ❶ DNA ࣐࢖ࢡࣟ࢔ࣞ࢖࡟ࡼࡿ⥙⨶ⓗ㑇ఏᏊⓎ⌧ゎᯒ

➨1 ⠇ ⥴ゝ ➨1 㡯 DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ  DNA ࣐࢖ࢡࣟ࢔ࣞ࢖(DNA ࢳࢵࣉ)࡜ࡣࠊ1 ᯛᩘ cm2 _{ࡢࢫࣛ࢖ࢻࢢࣛࢫ(2.5} cm×8.5 cm)࠿ࡽ࡞ࡿᇶᯈୖ࡟ࠊᩘⓒ࠿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ࢆᣢࡘDNA ࡸ┦ྠᛶࡢ 㧗࠸DNA ࡣ஫࠸࡟⤖ྜࡀྍ⬟࡛࠶ࡿࡀࠊ୍ḟᵓ㐀ࡀ␗࡞ࡿ DNA ࡣ⤖ྜࡍࡿࡇ ࡜ࡣ࡞࠸ࠋᚑࡗ࡚ࠊࢧࣥࣉࣝ୰࡟ᏑᅾࡍࡿDNA ࢳࢵࣉୖࡢࣉ࣮ࣟࣈ࡜┦ྠᛶࡢ 㧗࠸DNA ࡣࠊᩘ༓✀㢮ࡢ DNA ࡢ୰࡛ࡑࡢ┦ྠᛶࡢ㧗࠸ࣉ࣮ࣟࣈࢆ㑅ᢥࡋ࡚⤖ ྜࡍࡿࠋ  DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ࡣࠊ⣽⬊࠿ࡽᢳฟࡋࡓ mRNA ࢆ⏝࠸࡚ࠊᐇ㦂᮲௳࡟࠾ࡅ ࡿ㑇ఏᏊࡢⓎ⌧㔞ࢆゎᯒࡍࡿࠋࡑࡢࡓࡵ࡟DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒࡣࠊቑṪࣇ ࢙࢖ࢬࡸ⎔ቃࢫࢺࣞࢫࠊ໬Ꮫ≀㉁ࠊ✺↛ኚ␗➼ࡢせᅉ࡟ࡼࡗ࡚ᘬࡁ㉳ࡇࡉࢀࡿ㑇 ఏᏊⓎ⌧ࡢ⥙⨶ⓗ࡞ኚ໬ࢆ᫂ࡽ࠿࡟ࡍࡿࡇ࡜ࡀ࡛ࡁࡿࠋ

21

➨2 㡯 DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒࡢᛂ⏝౛

DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒࡣࠊྠ᫬࡟ᩘ༓✀㢮௨ୖࡢ㑇ఏᏊࡢⓎ⌧ኚ໬ࢆほᐹ ࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿࠋࡇࢀࡲ࡛࡟࢝ࢻ࣑࣒࢘(Momose and Iwahashi, 2001)ࠊ࢝ ࣉࢧ࢖ࢩࣥ(Kurita et al., 2002)ࠊ෾⤖(Odani et al., 2003)ࠊDMSO (Murata et al., 2003)ࠊ ࣛ࢘ࣥࢻ࢔ࢵࣉ(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 ࡓᚋࡢmRNA ࣉࣟࣇ࢓࢖ࣝࢆሗ࿌ࡋࡓࠋࡋ࠿ࡋࠊࡇࡢࡼ࠺࡞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

ࡇ࡜ࡣ㞴ࡋ࠸(Kobori et al., 1995)ࠋ㧗ᅽฎ⌮࡟ࡼࡿ⣽⬊࡬ࡢᙳ㡪ࢆ DNA ࣐࢖ࢡ ࣟ࢔ࣞ࢖࡛ホ౯ࡍࡿࡓࡵ࡟ࡣࠊ㧗ᅽฎ⌮࡟ࡼࡾ⏕ࡌࡓᦆയࢆᅇ᚟ࡍࡿ᫬࡟ኚ໬

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 ᅇ᚟ᇵ㣴ࡋࡓ⣽⬊࠿ࡽ RNA ࢆᢳฟࡋ࡚ 㑇ఏᏊⓎ⌧ࣉࣟࣇ࢓࢖ࣝࢆゎᯒࡋࡓ࡜ࡇࢁࠊ࢜ࣝ࢞ࢿࣛࡸ⭷ᵓ㐀࡟㛵㐃ࡍࡿࢱ

22 ࡢ⤖ᯝ࠿ࡽࠊ⮴Ṛⓗ࡞㧗ᅽ᮲௳࡟᭚ࡉࢀࡓ⣽⬊ࡣࠊ࢜ࣝ࢞ࢿࣛࡸ⭷ᵓ㐀➼ࡢಟ᚟ ࢆάⓎ࡟⾜ࡗ࡚࠸ࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࠊ㧗ᅽฎ⌮ࡣ࢜ࣝ࢞ࢿࣛࡸ⭷ᵓ㐀➼࡟ᦆ യࢆᘬࡁ㉳ࡇࡍࡇ࡜ࡀ♧၀ࡉࢀࡓࠋྠᵝ࡟㠀⮴Ṛⓗ࡞ᅽຊ࡬ࡢ㐺ᛂࢆゎᯒࡍࡿ ࡓࡵ࡟ࠊ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Ⓨ⌧ࡀ࢔ࢵࣉࣞࢠ࣮ࣗࣞ ࢺࡉࢀ࡚࠸ࡓࠋࡇࢀࡽࡢሗ࿌ࡣࠊDNA ࣐࢖ࢡࣟ࢔ࣞ࢖࡟ࡼࡾᅽຊ୙άᛶ໬࠶ࡿ ࠸ࡣ㧗ᅽ᮲௳࡬ࡢ㐺ᛂ࡜࠸࠺」㞧࡞ࣉࣟࢭࢫࡀゎᯒ࡛ࡁࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋ ➨3 㡯 DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ࡟࠾ࡅࡿࢡࣟࢫࣁ࢖ࣈࣜࢲ࢖ࢮ࣮ࢩࣙࣥ  DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒࡣࠊࠕ࠶ࡿ㑇ఏᏊࡣ┦ྠᛶࡢ㧗࠸㑇ఏᏊ࡜ࣁ࢖ࣈࣜ ࢲ࢖ࢬ(஺㞧ࠊ⤖ྜ)ࡍࡿࠖ(ᒾᶫ, 2002)࡜࠸࠺ཎ⌮࡟ᇶ࡙ࡃࠋᩘ༓✀㢮ࡶࡢ㑇ఏᏊ Ⓨ⌧ࢆྠ᫬࡟⥙⨶ⓗ࡟ゎᯒ࡛ࡁࡿ฼Ⅼࡀ࠶ࡿࡀࠊ┦ྠᛶࡀ㧗࠸ࡀ୍ḟᵓ㐀ࡢ␗ ࡞ࡿ㑇ఏᏊࢆࡶㄗࡗ࡚⤖ྜࡋ࡚ࡋࡲ࠺ࢡࣟࢫࣁ࢖ࣈࣜࢲ࢖ࢮ࣮ࢩࣙࣥࢆᘬࡁ㉳ ࡇࡍ(Iwahashi et al., 2007)ࠋ㑇ఏᏊⓎ⌧ࡢ⥙⨶ⓗゎᯒ࡟࠾࠸࡚୙☜ᐇᛶࢆᘬࡁ㉳ ࡇࡍࢡࣟࢫࣁ࢖ࣈࣜࢲ࢖ࢮ࣮ࢩࣙࣥࡣࠊDNA ࢳࢵࣉୖࡢࢃࡎ࠿ᩘ༑ሷᇶࡢࣉࣟ ࣮ࣈ࡟ࡼࡗ࡚ᩘ༓✀㢮ࡢ㑇ఏᏊࢆ㆑ูࡍࡿࡓࡵ࡟ᘬࡁ㉳ࡇࡉࢀࡿࣜࢫࢡ࡛࠶ࡿࠋ ࡋ࠿ࡋࠊ㑇ఏᏊⓎ⌧ࣉࣟࣇ࢓࢖ࣝ඲యࢆಠ▔ࡋࠊ᭷ព࡟Ⓨ⌧ࡀኚືࡍࡿ㑇ఏᏊ⩌

23 ࡢᶵ⬟ࢆホ౯ࡍࡿࡓࡵ࡟ࡣࠊ᭷⏝࡛࠶ࡿࠋᚑࡗ࡚ࠊDNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒࡣࠊ Ⓨ⌧ࡀኚ໬ࡋࡓ 1 㑇ఏᏊࡢࡳ࡟ὀ┠ࡍࡿࡢ࡛ࡣ࡞ࡃࠊⓎ⌧ࡢኚ໬ࡋࡓ㑇ఏᏊ⩌ ࡢᶵ⬟࡟╔┠ࡍࡿ࡭ࡁ࠶ࡿࠋࡲࡓࠊࡑࢀࡽࡢ㑇ఏᏊ⩌ࡢⓎ⌧ࡣࠊᚲせ࡟ᛂࡌ࡚ࢡ ࣟࢫࣁ࢖ࣈࣜࢲ࢖ࢮ࣮ࢩࣙࣥࡀᘬࡁ㉳ࡇࡉࢀ࡞࠸ࡼ࠺࡟ཝᐦ࡟タィࡉࢀࡓࣉࣛ ࢖࣐࣮ࢆ⏝࠸ࡓ㏫㌿෗PCR (RT-PCR)➼࡟ࡼࡗ࡚☜ㄆࡍࡿࡇ࡜ࡀ㔜せ࡛࠶ࡿࠋ ➨4 㡯 ᐇ㦂┠ⓗ  ᮏ❶࡛ࡣࠊᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴ࠾ࡼࡧࡑࡢぶᰴ KA31a ᰴࡢ㑇ఏᏊⓎ ⌧ࣉࣟࣇ࢓࢖ࣝࢆDNA ࣐࢖ࢡࣟ࢔ࣞ࢖࡟ࡼࡗ࡚ẚ㍑ࡍࡿࡇ࡜࡟ࡼࡾࠊᅽຊឤཷ ᛶ⬟ࢆ௜୚ࡍࡿ㑇ఏⓗせᅉࢆゎᯒࡋࡓࠋDNA ࣐࢖ࢡࣟ࢔ࣞ࢖ࡣࠊࢡࣟࢫࣁ࢖ࣈ ࣜࢲ࢖ࢮ࣮ࢩࣙࣥ࡟ࡼࡗ࡚ኚ␗㑇ఏᏊࡢྠᐃ࡟୙☜ᐇᛶࢆᘬࡁ㉳ࡇࡍࡀࠊ㑇ఏ ᏊⓎ⌧ࣉࣟࣇ࢓࢖ࣝࡢ᭷ព࡞ኚືࡣኚ␗㑇ఏᏊࡢᶵ⬟ࢆホ౯ࡍࡿࡓࡵ࡟ᙺ❧ࡘࠋ

24 ➨ ➨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 ⥺↷ᑕ࡟ࡼࡿ✺↛ኚ␗࡟ࡼࡾྲྀᚓࡋࡓ(Shigematsu et al., 2010a)ࠋࡇࢀࡽࡢ㓝ẕᰴ ࡣࠊ᪂₲⸆⛉኱Ꮫ 㔜ᯇ ஽ ᩍᤵࡼࡾศ୚ࡋ࡚࠸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᭤⥺ࡣHasegawa ࡽ(2012)ࡢ᪉ἲ࡟ᚑ࠸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(MX-301; Tomy Seiko, ᮾி, ᪥ᮏ)࡟ࡼࡾᅇ཰ࡋࡓࠋTotal RNA ࡣ Fast RNA® _{Pro Red}

25

Kit (MP Biomedicals, CA, USA)ࢆ⏝࠸࡚ࣇ࢙࣮ࣀ࣮ࣝ-ࢡ࣒ࣟࣟ࣍ࣝἲ࡟ࡼࡾᢳฟ ࡋࡓࠋ⣽⬊◚○ࢆMulti-Beads Shocker® _{(Ᏻ஭ჾᲔ, ኱㜰, ᪥ᮏ)࡛ 10 min ࡟ࠊࢡ}

࣒ࣟࣟ࣍ࣝฎ⌮ࢆ2 ᅇ࡟ኚ᭦ࡋࡓ௨እࡣᢳฟ࢟ࢵࢺࡢࣉࣟࢺࢥࣝ࡟ᚑࡗࡓࠋRNA ⃰ᗘ࠾ࡼࡧ⣧ᗘࡣ 260 nm ࡢ྾ගᗘ࠾ࡼࡧ Agilent 2100 BioanalyzerTM _(Agilent

Technologies, CA, USA)࡟ࡼࡾ ᐃࡋࡓࠋ

➨4 㡯 DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ᪉ἲ

DNA ࣐࢖ࢡࣟ࢔ࣞ࢖࡟ࡼࡿ㑇ఏᏊⓎ⌧ࡢ⥙⨶ⓗゎᯒࡣࠊYeast Oligo Microarray Kit (V2)TM _{(Agilent Technologies)ࢆ⏝࠸࡚ゎᯒࡋࡓࠋࡇࡢ࣐࢖ࢡࣟ࢔ࣞ࢖࡛ࡣࠊS.} cerevisiae S288c ᰴࡢ 6,256 㑇ఏᏊ࡟ᑐᛂࡍࡿ DNA ࣉ࣮ࣟࣈࢆ⏝࠸ࡓࠋ㑇ఏᏊⓎ

⌧ゎᯒࡣ 1 Ⰽἲ࡟ࡼࡾ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 ࡟ࡼࡿࣛ࣋ࣝ໬ࠊࣉ࣮ࣟࣈ࡜ࡢࣁ࢖ࣈࣜࢲ࢖ࢮ࣮ࢩࣙࣥࡣ Ecogenomics (⚟ ᒸ, ᪥ᮏ)࡟ጤクࡋࡓࠋ

26

➨5 㡯 㑇ఏᏊⓎ⌧ࡢศ㢮ゎᯒ᪉ἲ

ࡑࢀࡒࢀࡢORF(Open Reading Frame)࠿ࡽ᳨ฟࡉࢀࡓࢩࢢࢼࣝࡣࠊࢡ࢜ࣥࢱ࢖ ࣝἲ࡟ࡼࡾṇつ໬ࡋࡓࠋKA31a ᰴ᳨࡛ฟࡉࢀࡓࢩࢢࢼࣝ࡜ẚ࡭࡚ 2 ಸ௨ୖ࠶ࡿ ࠸ࡣ༙ศ௨ୗ࡟a924E1 ᰴࡢࢩࢢࢼࣝᙉᗘࡀኚ໬ࡋࡓ㑇ఏᏊࢆࡑࢀࡒࢀ࢔ࢵࣉࣞ ࢠ࣮ࣗࣞࢺ㑇ఏᏊࠊࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊ࡜ࡋࡓࠋྛ㑇ఏᏊࡣࢫࢳ࣮ࣗࢹࣥ

ࢺࡢt ᳨ᐃ(p < 0.05)࡟ࡼࡾ⤫ィฎ⌮ࡋࡓࠋⓎ⌧ࡀኚ໬ࡋࡓ㑇ఏᏊࡣ MIPS GenRE

CYGD database (http://mips.helmholtz-muenchen.de/genre/proj/yeast/)࡟ࡼࡾゎᯒࡋ ࡓࠋᮏ◊✲ࡢDNA ࣐࢖ࢡࣟ࢔ࣞ࢖ࢹ࣮ࢱࡣ MIAME ᇶ‽࡟‽ᣐࡋࠊ⏕ࢹ࣮ࢱࡣ Gene Expression Omnibus (GEO) database ࡟ᐤクࡋࡓ(GSE55120)ࠋ

➨6 㡯 quantitative PCR ᪉ἲ

ୖグࡢ᪉ἲ࡛㓝ẕ⣽⬊࠿ࡽᢳฟࡉࢀࡓtotal RNA ࡣࠊReverTra AceR qPCR RT Master Mix (TOYOBO, Osaka, Japan)ࢆ⏝࠸࡚ 37°Cࠊ15 min ࡢ᮲௳࡛㏫㌿෗ࡋࡓࠋ DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ࡢ⤖ᯝ࡟ᇶ࡙ࡁࠊ࢔ࢵࣉࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊ࠾ࡼࡧࢲ࢘ࣥ ࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊࡢࣉࣛ࢖࣐࣮ࢆ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Ⓨ

27

⌧ࣞ࣋ࣝࢆ⟬ฟࡋࡓࠋCDC48 㑇ఏᏊࡣ⣽⬊࿘ᮇ࡟㛵㐃ࡍࡿᚲ㡲㑇ఏᏊ࡛࠶ࡾࠊ

a924E1 ᰴ࠾ࡼࡧ KA31a ᰴࡢ୧ᰴ࡟࠾࠸࡚ྠ➼ࡢⓎ⌧㔞࡛࠶ࡗࡓࡓࡵ࡟ࣁ࢘ࢫ ࣮࢟ࣆࣥࢢ㑇ఏᏊ࡜ࡋ࡚฼⏝ࡋࡓࠋ

28

➨

➨3 ⠇ ⤖ᯝ

➨1 㡯 㑇ఏᏊⓎࣉࣟࣇ࢓࢖ࣝࡢᴫせ

ᮏ❶࡛ࡣࠊDNA ࣐࢖ࢡࣟ࢔ࣞ࢖࡟ࡼࡾᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴࡢ㑇ఏᏊ Ⓨ⌧ࣉࣟࣇ࢓࢖ࣝࢆゎᯒࡋࡓࠋTotal RNA ࡣ a924E1 ᰴ࠾ࡼࡧ KA31a ᰴ࠿ࡽࡑࢀ ࡒࢀ4 ᅇࡢ⊂❧ࡋࡓᐇ㦂࡟ࡼࡾᢳฟࡉࢀࡓࠋᢳฟ RNA ࡢ⃰ᗘࡣ࠾ࡼࡑ 1.2 μg/μL ࡛࠶ࡿࠋࡇࡢRNA ࢆ௨ୗࡢ DNA ࣐࢖ࢡࣟ࢔ࣞ࢖࠾ࡼࡧ RT-PCR ࡟౪ࡋࡓࠋ  DNA ࣐࢖ࢡࣟ࢔ࣞ࢖࡟ࡼࡾ᳨ฟࡉࢀࡓࢩࢢࢼࣝࢹ࣮ࢱࡣࢡ࢜ࣥࢱ࢖ࣝἲ࡟ࡼ 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᭷ព࡟ప࠿ࡗࡓ(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ఏᏊࡢ๭ྜࡀ᭱ࡶ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

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CYC7ࠊPET191ࠊSHY1ࠊNCA2ࠊCOQ8 㑇ఏᏊ)ࡸ࣑ࢺࢥࣥࢻࣜ࢔ࡢࣜ࣎ࢯ࣮࣒ྜ

ᡂ࡟㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊ➼ 45 㑇ఏᏊ(MRPL1ࠊMRPS35ࠊ

MBA1 㑇ఏᏊ)ࡀ࢔ࢵࣉࣞࢠ࣮ࣗࣞࢺࡉࢀ࡚࠸ࡓࠋ

 “Biogenesis of cellular components”࢝ࢸࢦ࣮ࣜࡣ࢔ࢵࣉࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊࡢ ๭ྜࡀ2 ␒┠࡟ከ࠿ࡗࡓ(⾲ 3)ࠋྠ࢝ࢸࢦ࣮ࣜ࡟ศ㢮ࡉࢀࡿ 862 㑇ఏᏊࡢ࠺ࡕ 88 㑇ఏᏊࡀ࢔ࢵࣉࣞࢠ࣮ࣗࣞࢺࡉࢀ࡚࠸ࡓࠋࡲࡓࠊྠ࢝ࢸࢦ࣮ࣜࡢୗ఩࢝ࢸࢦ࣮ࣜ ࡛࠶ࡿ“Mitochondrion”ࢧࣈ࢝ࢸࢦ࣮ࣜ࡟ศ㢮ࡉࢀࡿ 171 㑇ఏᏊࡢ࠺ࡕ 32 㑇ఏᏊ ࡀ࢔ࢵࣉࣞࢠ࣮ࣗࣞࢺࡉࢀ࡚࠸ࡓࠋࡑࢀࡽࡢ࢔ࢵࣉࣞࢠ࣮ࣗࣞࢺࡉࢀࡓ㑇ఏᏊ ࡢ࠺ࡕࠊ21 㑇ఏᏊࡀ࣑ࢺࢥࣥࢻࣜ࢔ࣜ࣎ࢯ࣮࣒࡟㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻ ࡍࡿ㑇ఏᏊ(MRP51ࠊMRPL1ࠊMRPS35ࠊRSM25ࠊVAR1 㑇ఏᏊ)࡛࠶ࡗࡓࠋ  ࡑࢀࡒࢀࡢ㑇ఏᏊࡀࢥ࣮ࢻࡍࡿࢱࣥࣃࢡ㉁ࡢᒁᅾ఩⨨ศ㢮(⾲ 4)࡟࠾࠸࡚ࡣࠊ ࣑ࢺࢥࣥࢻࣜ࢔࡟ᒁᅾࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ“Mitochondria”࢝ࢸࢦ࣮ࣜ࡟ ศ㢮ࡉࢀࡿ㑇ఏᏊ⩌ࡀ᭱ࡶప࠸ p ್ࢆ♧ࡋࡓ(p < 0.01)ࠋࡇࢀࡽࡢ࢔ࢵࣉࣞࢠࣗ ࣮ࣞࢺࡉࢀࡓ㑇ఏᏊࡀࢥ࣮ࢻࡋ࡚࠸ࡿࢱࣥࣃࢡ㉁ࡣ㓟໬ⓗࣜࣥ㓟໬࡟㛵୚ࡋ࡚ ࠾ࡾࠊ࢚ࢿࣝࢠ࣮⏘⏕࡟㔜せ࡛࠶ࡿࠋࡇࢀࡽࡢ⤖ᯝࡣࠊa924E1 ᰴ࡟࠾࠸࡚ᅽຊ ឤཷᛶኚ␗ᑟධ࡟ࡼࡾ࣑ࢺࢥࣥࢻࣜ࢔ᶵ⬟࡟㛵㐃ࡍࡿ㑇ఏᏊ࡟ᙳ㡪ࡀ⾲ࢀ࡚࠸ ࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋ

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➨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 ࢦ࣮ࣜ࡟ศ㢮ࡉࢀࡿ㑇ఏᏊ࡟࠾࠸࡚ኚື㑇ఏᏊࡢ㧗࠸๭ྜࠊప࠸ p ್ࢆ♧ࡋࡓ (⾲ 5)ࠋ“Protein synthesis”࢝ࢸࢦ࣮ࣜ࡟ศ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 ࢸࢦ࣮ࣜ࡟ศ㢮ࡉࢀࡓࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊࡢ࠸ࡃࡘ࠿ࡣࠊࣜ࣎ࢯ࣮࣒ࡢ ⏕ྜᡂࡸrRNA ࡢྜᡂࠊࣉࣟࢭࢵࢩࣥࢢࠊಟ㣭➼࡟㛵㐃ࡍࡿࠋࡇࢀࡽࡢ㑇ఏᏊࡀ ࢥ࣮ࢻࡍࡿࢱࣥࣃࢡ㉁ࡣ୺࡟“Cytoplasm”࠾ࡼࡧ“Nucleus”࡟ᒁᅾࡋ࡚࠸ࡓ(⾲ 6)ࠋ ࡲࡓࠊࣂࣜࣥࠊࣟ࢖ࢩࣥࠊ࢖ࢯࣟ࢖ࢩࣥࠊ࢔ࣝࢠࢽࣥࠊࢭࣜࣥࠊࢺࣜࣉࢺࣇ࢓ࣥ ➼ࡢ࢔࣑ࣀ㓟ࡢ⏕ྜᡂ࡟㛵㐃ࡍࡿࢧࣈ࢝ࢸࢦ࣮ࣜ࡟ศ㢮ࡉࢀࡓ㑇ఏᏊࡶࢲ࢘ࣥ ࣞࢠ࣮ࣗࣞࢺࡋࡓࠋࡇࢀࡽࡢ⤖ᯝࡣࠊኚ␗ᰴ࡟࠾࠸࡚ࢱࣥࣃࢡ㉁࠾ࡼࡧ࢔࣑ࣀ㓟 ࡢ⏕ྜᡂ࡟㛵୚ࡍࡿάᛶࡀῶᑡࡋ࡚࠸ࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋ

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➨4 㡯 quantitative PCR ࡟ࡼࡿ㑇ఏᏊⓎ⌧ゎᯒࡢホ౯

࢔ࢵࣉࣞࢠ࣮ࣗࣞࢺ࠾ࡼࡧࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊࡢⓎ⌧ࢆ☜ㄆࡍࡿࡓࡵ

࡟ྛ㑇ఏᏊ࡟ᑐᛂࡍࡿࣉࣛ࢖࣐࣮ࢆタィࡋࡓ(⾲ 2)ࠋquantitative PCR ࡟ࡼࡾ a924E1 ᰴ࠾ࡼࡧ KA31a ᰴࡢ㑇ఏᏊⓎ⌧ࢆホ౯ࡋࡓ(⾲ 7)ࠋa924E1 ᰴࡢ࢔ࢵࣉࣞ ࢠ࣮ࣗࣞࢺࡋࡓ㑇ఏᏊࡢ┦ᑐⓎ⌧ࣞ࣋ࣝࡣࠊKA31a ᰴࡢ┦ᑐⓎ⌧ࣞ࣋ࣝࡼࡾࡶ 2 ಸ௨ୖ㧗࠿ࡗࡓࠋ୍᪉ࠊa924E1 ᰴࡢࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺ㑇ఏᏊࡢ┦ᑐⓎ⌧ࣞ ࣋ࣝࡣKA31a ᰴ࡜ẚ㍑ࡋ࡚༙ศ௨ୗ࡛࠶ࡗࡓࠋࡇࢀࡽࡢ⤖ᯝࡣࠊDNA ࣐࢖ࢡࣟ ࢔ࣞ࢖࡟ࡼࡿ㑇ఏᏊⓎ⌧ゎᯒ⤖ᯝࡢጇᙜᛶࢆᨭᣢࡋ࡚࠸ࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊDNA ࣐࢖ࢡࣟ࢔ࣞ࢖࡟ࡼࡗ࡚࢔ࢵࣉࣞࢠ࣮ࣗࣞࢺࡉࢀ࡚࠸ࡓa924E1 ᰴࡢ COX1 㑇ఏ Ꮚࡢ┦ᑐⓎ⌧ࣞ࣋ࣝࡣ࡜࡚ࡶᑡ࡞࠿ࡗࡓࠋCOX1 㑇ఏᏊࡢูࡢ㡿ᇦࢆࢱ࣮ࢤࢵࢺ ࡜ࡋࡓࣉࣛ࢖࣐࣮࡛☜ㄆࡋࡓࡀࠊⓎ⌧ࢆほᐹࡍࡿࡇ࡜ࡣ࡛ࡁ࡞࠿ࡗࡓ(⾲ 7)ࠋ

32 ➨ ➨4 ⠇ ⪃ᐹ ᮏ❶࡛ࡣࠊᅽຊឤཷᛶ⬟ࡢ௜୚ᶵᵓࢆ᫂ࡽ࠿࡜ࡍࡿࡓࡵ࡟ࠊDNA ࣐࢖ࢡࣟ࢔ ࣞ࢖࡟ࡼࡾᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴࡢ㑇ఏᏊⓎ⌧ࣉࣟࣇ࢓࢖ࣝࢆࡑࡢぶᰴ KA31a ᰴࡢࡶࡢ࡜ẚ㍑ࡋࡓࠋࡑࡢ⤖ᯝࠊa924E1 ᰴࡢ 1,000 ௨ୖࡢ㑇ఏᏊࡢⓎ⌧ ࡀ᭷ព࡟ኚ໬ࡋࡓࡇ࡜ࡀ᫂ࡽ࠿࡜࡞ࡗࡓ(p < 0.05)ࠋ≉࡟࢚ࢿࣝࢠ࣮௦ㅰᶵ⬟࡟ 㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢⓎ⌧ࡀ࢔ࢵࣉࣞࢠ࣮ࣗࣞࢺࡋ(⾲ 3)ࠊ ࢔࣑ࣀ㓟ࡸࢱࣥࣃࢡ㉁ࡢ⏕ྜᡂ࡟㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡢⓎ ⌧ࡀࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺࡋࡓ(⾲ 5)ࠋ࢔ࢵࣉࣞࢠ࣮ࣗࣞࢺࡋࡓ㑇ఏᏊࡢከࡃࡣ࣑ ࢺࢥࣥࢻࣜ࢔࡟ᒁᅾࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊ࡛࠶ࡗࡓࠋDNA ࣐࢖ࢡ ࣟ࢔ࣞ࢖࡟ࡼࡿ㑇ఏᏊⓎ⌧ࡢゎᯒ࡟࠾࠸࡚ὀពࡋ࡞ࡅࢀࡤ࡞ࡽ࡞࠸ࡇ࡜ࡣࠊⓎ ⌧ࡀኚືࡋࡓᶵ⬟㑇ఏᏊ⩌ࡀࠊ࡞ࡐⓎ⌧ࢆኚືࡉࡏࡿᚲせࡀ࠶ࡗࡓࡢ࠿DNA ࣐ ࢖ࢡࣟ࢔ࣞ࢖ゎᯒࡢ⤖ᯝ඲యࢆಠ▔ⓗ࡟⪃ᐹࡍࡿࡇ࡜࡛࠶ࡿࠋ࣑ࢺࢥࣥࢻࣜ࢔ ࡟㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡍࡿ㑇ఏᏊࡀ࢔ࢵࣉࣞࢠ࣮ࣗࣞࢺࡉࢀࡓࡇ࡜࠿ ࡽࠊa924E1 ᰴ࡟࠾࠸࡚࣑ࢺࢥࣥࢻࣜ࢔ᶵ⬟ࡀ㔜せ࡛࠶ࡿࡇ࡜ࡀ⪃࠼ࡽࢀࡿࠋࡑ ࡢཎᅉ࡜ࡋ࡚ࡣࠊᅽຊឤཷᛶኚ␗ࡢᑟධ࡟ࡼࡾձ࣑ࢺࢥࣥࢻࣜ࢔ࡀศゎ࠶ࡿ࠸ ࡣᶵ⬟୙඲࡜࡞ࡗࡓࡢ࠿ࠊղ࣑ࢺࢥࣥࢻࣜ࢔ᶵ⬟ࡀᙉ໬ࡉࢀࡓࡢ࠿ࢃ࠿ࡽ࡞࠸ࠋ ࡇࡢ⤖ᯝ࡜ేࡏ࡚ࠊࢲ࢘ࣥࣞࢠ࣮ࣗࣞࢺࡋࡓ㑇ఏᏊ⩌ࡀ࢔࣑ࣀ㓟ࡸࢱࣥࣃࢡ㉁

33 ࡢ⏕ྜᡂ࡟㛵㐃ࡍࡿࢱࣥࣃࢡ㉁ࢆࢥ࣮ࢻࡋ࡚࠸ࡓࡇ࡜࠿ࡽࠊᅽຊឤཷᛶ⬟ࡣ a924E1 ᰴࡢ࣑ࢺࢥࣥࢻࣜ࢔ᶵ⬟ࡀపୗࡍࡿࡼ࠺࡞ᙳ㡪ࢆᘬࡁ㉳ࡇࡋ࡚࠸ࡿྍ⬟ ᛶࡀ⪃࠼ࡽࢀࡿࠋ RT-PCR ࡟ࡼࡾ㑇ఏᏊⓎ⌧ࢆホ౯ࡋࡓ⤖ᯝࠊa924E1 ᰴࡢ࢔ࢵࣉࣞࢠ࣮ࣗࣞࢺ ࡋࡓ㑇ఏᏊࡢ┦ᑐⓎ⌧ࣞ࣋ࣝࡣࠊKA31a ᰴࡢ┦ᑐⓎ⌧ࣞ࣋ࣝࡼࡾࡶ 2 ಸ௨ୖ㧗 ࠸್ࢆ♧ࡋࡓ(⾲ 7)ࠋࡋ࠿ࡋ࡞ࡀࡽࠊDNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ࡟ࡼࡾ࢔ࢵࣉࣞࢠ ࣮ࣗࣞࢺࡋ࡚࠸ࡓ࣑ࢺࢥࣥࢻࣜ࢔ࡢ㓟໬ⓗࣜࣥ㓟໬࡟㛵㐃ࡍࡿࢩࢺࢡ࣒ࣟ c 㓟 ໬㓝⣲ࢆࢥ࣮ࢻࡍࡿCOX1 㑇ఏᏊࡢⓎ⌧ࡣ☜ㄆ࡛ࡁ࡞࠿ࡗࡓ(⾲ 7)ࠋ DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ࡣ DNA ࢳࢵࣉୖࡢࢃࡎ࠿ᩘ༑ሷᇶࡢࣉ࣮ࣟࣈ࡟ࡼࡗ࡚ ᩘ༓ࡢ㑇ఏᏊࢆ㆑ูࡍࡿ᪉ἲ࡛࠶ࡿࠋࢡࣟࢫࣁ࢖ࣈࣜࢲ࢖ࢮ࣮ࢩࣙࣥࡍࡿࡇ࡜ ࡀ↓࠸ࡼ࠺࡟ཝᐦ࡟タィࡉࢀࡓPCR ⏝ࡢࣉࣛ࢖࣐࣮࡜␗࡞ࡾࠊDNA ࣐࢖ࢡࣟ࢔ ࣞ࢖ࡢࣉ࣮ࣟࣈࡣࢡࣟࢫࣁ࢖ࣈࣜࢲ࢖ࢮ࣮ࢩࣙࣥࡍࡿࣜࢫࢡࡼࡾࡶᩘ༓✀㢮௨ ୖࡢ㑇ఏᏊࡢⓎ⌧ࢆ⥙⨶ⓗ࡟ゎᯒࡍࡿࡇ࡜ࢆඃඛࡋ࡚࠸ࡿࠋࡑࡢࡓࡵ࡟ࠊࡇࡢぢ ࠿ࡅୖࡢCOX1 㑇ఏᏊࡢ࢔ࢵࣉࣞࢠ࣮ࣗࣞࢩࣙࣥࡣࠊDNA ࣐࢖ࢡࣟ࢔ࣞ࢖࡟≉ ᭷ࡢࢡࣟࢫࣁ࢖ࣈࣜࢲ࢖ࢮ࣮ࢩ࡛ࣙࣥ࠶ࡿ࡜⪃࠼ࡽࢀࡿࠋDNA ࣉ࣮ࣟࣈࡢሷᇶ 㓄ิࡢ┦ྠᛶࡸ⺯ගᙉᗘࡢࡤࡽࡘࡁࠊ⣽⬊ࡢᇵ㣴᮲௳➼ࡀཎᅉ࡛ࡋࡤࡋࡤࢡࣟ ࢫࣁ࢖ࣈࣜࢲ࢖ࢮ࣮ࢩࣙࣥࡀᘬࡁ㉳ࡇࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Iwahashi et

34 ࡵ࡚㧗࠸┦ྠᛶࢆ♧ࡋࡓࠋ౛࠼ࡤࠊCOX1 㑇ఏᏊࣉ࣮ࣟࣈࡢ 40-60 bp 㒊ศࡣࠊ5-࣍ࢫ࣍ࣜ࣎ࢩࣝ-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㇟࡜ࡋࡓquantitative PCR ࡛෌ゎᯒࡋࡓ࡜ࡇࢁࠊⓎ⌧ࡣ☜ㄆ࡛ࡁ࡞࠿ࡗࡓ(⾲ 7)ࠋ ࡇࢀࡽࡢ⤖ᯝࡣࠊa924E1 ᰴ࡟࠾࠸࡚ COX1 㑇ఏᏊࡢⓎ⌧ࡀ࢔ࢵࣉࣞࢠ࣮ࣗࣞࢺ ࡋ࡚࠸࡞࠸ࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋ  ᮏ❶ࡢ⤖ᯝࢆࡲ࡜ࡵࡿ࡜ࠊa924E1 ᰴࡣᅽຊឤཷᛶኚ␗ࡢᑟධ࡟ࡼࡾ࣑ࢺࢥࣥ ࢻࣜ࢔ࡸ࢔࣑ࣀ㓟➼ࡢࢱࣥࣃࢡ㉁ࡢ⏕ྜᡂ࡟㛵㐃ࡍࡿᶵ⬟࡟ᙳ㡪ࡀ⏕ࡌࡓࡇ࡜ ࡀ♧၀ࡉࢀࡓࠋ࣑ࢺࢥࣥࢻࣜ࢔ࡣࠊᵝࠎ࡞࢔࣑ࣀ㓟➼ࡢ୰㛫௦ㅰ⏘≀ࡢ⏕ྜᡂࡢ ୺せ࡞௦ㅰ⤒㊰࡛࠶ࡾࠊ㔜せ࡞ᙺ๭ࢆᢸ࠺ჾᐁ࡛࠶ࡿࠋ➨3 ❶࡛ࡣࠊDNA ࣐࢖ ࢡࣟ࢔ࣞ࢖ゎᯒ࡟ᇶ࡙ࡁࠊa924E1 ᰴࡢᅽຊឤཷᛶ⬟ࡀཬࡰࡍ࣑ࢺࢥࣥࢻࣜ࢔ᶵ ⬟࡬ࡢᙳ㡪࡟ࡘ࠸࡚ゎᯒࡍࡿࠋ

35

➨

➨

3 ❶ ࣑ࢺࢥࣥࢻࣜ࢔ᶵ⬟ࡢゎᯒ



 ➨1 ⠇ ⥴ゝ ➨1 㡯 㓝ẕ࡜࣑ࢺࢥࣥࢻࣜ࢔  ࣑ࢺࢥࣥࢻࣜ࢔ࡣ㓟໬ⓗࣜࣥ㓟໬࡟ࡼࡿ࢚ࢿࣝࢠ࣮⏕⏘ࢆྖࡿ⣽⬊ᑠჾᐁ࡛ ࠶ࡿࠋ࣑ࢺࢥࣥࢻࣜ࢔ࡣ⊂⮬࡟DNA(࣑ࢺࢥࣥࢻࣜ࢔ DNA)ࢆᣢࡕࠊศ⿣ࠊቑṪ ࢆ⾜࠺ࡇ࡜ࡀ࡛ࡁࡿࠋ㓝ẕࡢ࣑ࢺࢥࣥࢻࣜ࢔DNA ࡣ࠾ࡼࡑ 76 kbp ࡛࠶ࡾࠊࣄࢺ ࡣ࠾ࡼࡑ16.5 kbp ࡛࠶ࡿ(⚟ཎ, 1986)ࠋ࡯࡜ࢇ࡝ࡢ┿᰾⏕≀ࡣ࢚ࢿࣝࢠ࣮⋓ᚓࢆ ዲẼ࿧྾࡟౫Ꮡࡋ࡚࠸ࡿࡓࡵ࡟ࠊ࣑ࢺࢥࣥࢻࣜ࢔DNA ࡢḞᦆ➼࡟㉳ᅉࡍࡿ࣑ࢺ ࢥࣥࢻࣜ࢔ᶵ⬟ࡢపୗࡣ⮴࿨ⓗ࡛࠶ࡿࠋ࣑ࢺࢥࣥࢻࣜ࢔DNA ࡢḞᦆ࡟㛵㐃ࡋ࡚ ࠸ࡿࣄࢺࡢ㑇ఏ⑓ࡶᑡ࡞ࡃ࡞࠸ࠋࡲࡓࠊ࣑ࢺࢥࣥࢻࣜ࢔ࡢ㓟໬ⓗࣜࣥ㓟໬࡟ࡼࡾ ⏕ᡂࡉࢀࡿάᛶ㓟⣲ࡀ⒴ࢆᘬࡁ㉳ࡇࡍࡇ࡜ࡶ♧၀ࡉࢀ࡚࠸ࡿࠋ  㓝ẕࡣ࢚ࢱࣀ࣮ࣝⓎ㓝࡟ࡼࡾ⏕Ꮡࡍࡿࡇ࡜ࡀྍ⬟࡛࠶ࡿࡓࡵࠊ࣑ࢺࢥࣥࢻࣜ ࢔DNA ࡢḞᦆࡣࣄࢺ࡯࡝⮴࿨ⓗ࡛ࡣ࡞࠸ࠋᐇ㝿࡟ Ephrussi ࡽ(1955)ࡶሗ࿌ࡋ࡚ ࠸ࡿࡼ࠺࡟㓝ẕࡢ࿧྾Ḟᦆᰴ࡟ࡘ࠸࡚ࡣᩘከࡃሗ࿌ࡉࢀ࡚࠾ࡾࠊ≉࡟࣑ࢺࢥࣥ

ࢻࣜ࢔ DNA ࡀḞኻࡋࡓᰴࢆ rho㸫_{ᰴࠊ᏶඲࡟Ḟኻࡋࡓᰴࢆ rho}0_{ᰴ࡜࿧ࡪࠋ㓝ẕ}

36 ࡃ᭷ࡋ࡚࠸ࡿࠋࡑࡢࡓࡵ࡟࣑ࢺࢥࣥࢻࣜ࢔ࡀ㛵㐃ࡍࡿ㑇ఏ⑓ࡸ⒴ࢆጞࡵ࡜ࡋࡓ ከࡃࡢ⑓Ẽࡸ࿧྾㜼ᐖ๣➼࡟ᑐࡍࡿᇶ♏◊✲࡜ࡋ࡚㓝ẕࡢ࣑ࢺࢥࣥࢻࣜ࢔◊✲ ࡀάⓎ࡟㐍ࡵࡽࢀ࡚࠸ࡿ (⚟ཎ, 1986)ࠋ ➨2 㡯 ᐇ㦂┠ⓗ  ➨ 2 ❶ࡢ DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ࡟ࡼࡾࠊa924E1 ᰴࡢᅽຊឤཷᛶ࡜࣑ࢺࢥ ࣥࢻࣜ࢔ࡢ㛵㐃ᛶࡀ♧၀ࡉࢀࡓࠋ࣑ࢺࢥࣥࢻࣜ࢔ࡣ࿧྾ᶵ⬟ࡸ⣽⬊ෆ௦ㅰࡢ୰ 㛫௦ㅰ⏘≀ࢆ⏕⏘ࡍࡿ㔜せ࡞ჾᐁ࡛࠶ࡿࠋᮏ❶࡛ࡣ࣑ࢺࢥࣥࢻࣜ࢔ᶵ⬟ࢆ⾲⌧ ᙧ㉁ゎᯒ࡟ࡼࡾホ౯ࡋࡓࠋ

37 ➨ ➨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 ஽ ᩍᤵࡼࡾศ୚ࡋ࡚࠸ࡓࡔ࠸ࡓࠋࡲࡓࠊ➨ 3 㡯࡛సฟࡋࡓ஧ಸయᰴࢆ౑⏝ 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)ࡢ᪉ἲ࡟ᚑ࠸ࠊBio Microplate Reader HiTSTM_{ࢆ⏝࠸࡚30°Cࠊ120 h ࡢ᮲௳}

࡛ ᐃࡋࡓࠋ

➨3 㡯 ஧ಸయᰴࡢసฟ

 ᮏᐇ㦂࡛౑⏝ࡋࡓ஧ಸయᰴࡣࠊᅽຊឤཷᛶኚ␗ᰴ a924E1 ᰴ(a ᆺ୍ಸయᰴ)࡜ 㔝⏕ᆺᰴKA31α ᰴ(α ᆺ୍ಸయᰴ)ࡢ᥋ྜ࡟ࡼࡾసฟࡋࡓࠋ୧ᰴࡢᇵ㣴ᾮ 300 μL ࢆYPD ᇵᆅ 2.4 mL ࡟ຍ࠼࡚ࡼࡃΰྜࡋࠊ30°Cࠊ24 h ࡢ᮲௳࡛㟼⨨ᇵ㣴ࡋࡓࠋᇵ

38

㣴ᚋࠊ఩┦ᕪ㢧ᚤ㙾࡟ࡼࡾ᥋ྜᏊࢆ☜ㄆࡋ࡚YPD ᐮኳᇵᆅୖ࡟᥋ྜ⣽⬊ࢆ࢔࢖ ࢯ࣮ࣞࢩࣙࣥࡋࠊ༢㞳ࡋࡓࢥࣟࢽ࣮ࢆ஧ಸయೃ⿵ᰴ࡜ࡋࡓࠋ஧ಸయೃ⿵ᰴࢆ෌ࡧ

a924E1 ᰴ࠾ࡼࡧ KA31α ᰴࡢᇵ㣴ᾮ࡜ࡑࢀࡒࢀΰྜࡋࠊ᥋ྜᏊࡀほᐹࡉࢀ࡞࠿ࡗ ࡓࢥࣟࢽ࣮ࢆ஧ಸయᰴ࡜ࡋࡓࠋసฟࡋࡓ஧ಸయᰴࡣࠊFACS CaliburTM flow cytometer (Becton Dickinson and Co., NJ, ⡿ᅜ)࡟ࡼࡾ DNA 㔞࠾ࡼࡧ⣽⬊ࢧ࢖ࢬࢆ ☜ㄆࡋࡓࠋ

➨4 㡯 ࿧྾ᶵ⬟ࡢゎᯒ᪉ἲ

 㓝ẕᰴࡢ࿧྾ᶵ⬟ࡣ 2,3,5-triphenyl-2H-tetrazolium chloride (TTC; Wako Pure Chemical Industries)࡟ࡼࡿࢥࣟࢽ࣮ᰁⰍἲ࡟ࡼࡾ Nagai(1959)ࡢሗ࿌࡟ᚑ࠸ゎᯒ ࡋࡓࠋ

➨5 㡯 ࣑ࢺࢥࣥࢻࣜ࢔ DNA Ḟኻࡢゎᯒ᪉ἲ

࣑ࢺࢥࣥࢻࣜ࢔ DNA ࡢḞኻࡣ PCR ἲ࡟ࡼࡾゎᯒࡋࡓࠋTotal DNA ࡣࠊDr. GenTLE® _{(from Yeast) High Recovery (ࢱ࢝ࣛࣂ࢖࢜)ࢆ⏝࠸ࡓࢨ࢖ࣔࣜ࢔࣮ࢮฎ⌮}

࡟ࡼࡾᢳฟࡋࡓࠋᢳฟࡋࡓDNA ࡣ TE ࣂࢵࣇ࢓࣮50 μL ࡟⁐ゎࡋࠊ㸫20°C ࡛ಖ ᏑࡋࡓࠋDNA ⃰ᗘࡣ 260 nm ࡢ྾ගᗘ࡛ ᐃࡋࡓࠋ࣑ࢺࢥࣥࢻࣜ࢔ DNA ࡢቑᖜ ࡢࡓࡵ࡟ࠊSaccharomyces Genome Database (SGD, http://www.yeastgenome.org/) ࠾

39 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ఏᏊ࡜ࡋ࡚ࡣ CDC48 㑇ఏᏊࢆ⏝ ࠸ࡓࠋ ➨6 㡯 㧗ᅽฎ⌮᪉ἲ

㧗ᅽฎ⌮ࡣࠊShigematsu ࡽ(2010a)ࡢሗ࿌࡟ᚑ࠸ࠊ200 MPaࠊ20°Cࠊ0-360 s ࡢ᮲ ௳࡛⾜ࡗࡓࠋ

40 ➨ ➨3 ⠇ ⤖ᯝ ➨1 㡯 ࿧྾ᶵ⬟ࡢゎᯒ DNA ࣐࢖ࢡࣟ࢔ࣞ࢖ࡢ⥙⨶ⓗ㑇ఏᏊⓎ⌧ゎᯒࡢ⤖ᯝ࠿ࡽ a924E1 ᰴ࡟࠾࠸࡚ࠊ ࣑ࢺࢥࣥࢻࣜ࢔ࡢᶵ⬟ࡀᅽຊឤཷᛶ⬟࡟ᙳ㡪ࢆ୚࠼࡚࠸ࡿࡇ࡜ࡀ♧၀ࡉࢀࡓࠋ ࡑࡇ࡛ TTC ᰁⰍἲ࡟ࡼࡾ a924E1 ᰴࡢ࣑ࢺࢥࣥࢻࣜ࢔ࡢ࿧྾ᶵ⬟ࢆホ౯ࡋࡓࠋ ↓Ⰽࡢࢸࢺࣛࢰ࣒ࣜ࢘ሷࡣ㟁Ꮚཷᐜయ࡜ࡋ࡚ാࡁࠊ㓟໬ⓗࣜࣥ㓟໬ࡀ⾜ࢃࢀࡿ ࣑ࢺࢥࣥࢻࣜ࢔⭷㛫⭍࡛㉥Ⰽࡢ࣐࣍ࣝࢨࣥⰍ⣲࡟㑏ඖࡉࢀࡿࠋKA31a ᰴࡢࢥࣟ ࢽ࣮ࡣTTC ᐮኳᇵᆅࢆ㔜ᒙᚋ 20 min ࡛ࣆࣥࢡⰍ࡟ᰁࡲࡾࡣࡌࡵࠊ100 min ᚋ࡟ ࡣ⃰㉥Ⰽ࡟ᰁⰍࡉࢀࡓ(ᅗ 1)ࠋᑐ↷ⓗ࡟ࠊa924E1 ᰴࡢࢥࣟࢽ࣮ࡣ 100 min ࡀ⤒㐣 ࡋ࡚ࡶࢃࡎ࠿࡟ࣆࣥࢡⰍ࡟Ⰽ௜ࡃࡔࡅ࡛࠶ࡗࡓ(ᅗ 1)ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊa924E1 ᰴࡢዲẼ࿧྾⬟ࡢάᛶࡀపୗࡋ࡚࠸ࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࠋ ➨2 㡯 ࣑ࢺࢥࣥࢻࣜ࢔ DNA ࡢḞኻ 㓝ẕ࡟࠾ࡅࡿ࿧྾ᶵ⬟ࡢάᛶపୗࡣࠊ࣑ࢺࢥࣥࢻࣜ࢔DNA ࡢḞኻࡀཎᅉ࡛࠶ ࡿࡇ࡜ࡀከ࠸ࠋa924E1 ᰴࡢ࿧྾ᶵ⬟ࡢάᛶపୗࢆᘬࡁ㉳ࡇࡋࡓ㑇ఏⓗせᅉࢆྠ ᐃࡍࡿࡓࡵ࡟ࠊ࣑ࢺࢥࣥࢻࣜ࢔DNA ࢆᑐ㇟࡜ࡋ࡚ PCR ἲ࡟ࡼࡾゎᯒࡋࡓࠋ࣑ ࢺࢥࣥࢻࣜ࢔DNA ࡢ୺せ࡞㑇ఏᏊ࡛࠶ࡿ 15S rRNAࠊ21S rRNAࠊCOX1ࠊCOX3ࠊ

41

COB 㑇ఏᏊࢆࢱ࣮ࢤࢵࢺ࡜ࡋ࡚ࣉࣛ࢖࣐࣮ࢆ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ఏᏊࡢḞኻ࡜ᅽຊឤཷᛶ⬟ࡢ㛵㐃ᛶࢆゎᯒࡍࡿࡓࡵ࡟ࠊa ᆺ୍ಸయᰴ a924E1 ᰴ࠾ࡼࡧ α ᆺ୍ಸయ㔝⏕ᆺᰴ KA31α ᰴࡢ᥋ྜ࡟ࡼࡾ 5 ᰴࡢ஧ಸయᰴࢆ సฟࡋࡓࠋసฟࡋࡓ஧ಸయᰴࡢDNA 㔞࠾ࡼࡧ⣽⬊ࢧ࢖ࢬࡣࣇ࣮ࣟࢧ࢖ࢺ࣓ࢺࣜ ࣮࡛☜ㄆࡋࡓ(ᅗ 2)ࠋ஧ಸయᰴࡢ DNA 㔞(G1 ᮇ㸸2CࠊG2 ᮇ㸸4C)ࡣࠊ୍ಸయᰴ (G1 ᮇ㸸1CࠊG2 ᮇ㸸2C)ࡢ࠾ࡼࡑ 2 ಸ㔞࡛࠶ࡗࡓࠋ஧ಸయᰴࡢ G2 ᮇ(DNA 㔞㸸 4C)ࡢ⣽⬊ࡢࢧ࢖ࢬࡣࠊG1 ᮇ(2C)ࡢ⣽⬊ࡢ 2 ಸ௨ୖ࡛࠶ࡾࠊྠᵝ࡟୍ಸయᰴ࡟ ࠾࠸࡚ࡶࠊG2 ᮇ(DNA 㔞㸸2C)ࡢ⣽⬊ࡢࢧ࢖ࢬࡣࠊG1 ᮇ(1C)ࡢ⣽⬊ࡢ 2 ಸ௨ୖ ࡛࠶ࡗࡓࠋ஧ಸయᰴࡢቑṪ᭤⥺ࡣᅗ3 ࡟♧ࡍࠋ

42

➨4 㡯 ᅽຊ୙άᛶ໬ᣲືࡢゎᯒ

 సฟࡋࡓ஧ಸయᰴ࠾ࡼࡧa924E1 ᰴࠊKA31a ᰴࢆ 200 MPaࠊ20°Cࠊ0-360 s ࡢ᮲ ௳࡛㧗ᅽฎ⌮ࡋࡓࠋYPD ᐮኳᇵᆅ࡟ࡼࡿࢥࣟࢽ࣮࢝࢘ࣥࢺἲ࡟ࡼࡾ⏕⳦ᩘ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࡟ࣉࣟࢵࢺࡋ࡚ᅽຊ୙άᛶ໬᭤⥺ࢆసᡂࡋࠊᅽຊ୙ άᛶ໬㏿ᗘᐃᩘࢆ⟬ฟࡋࡓ(ᅗ 4)ࠋࡑࡢ⤖ᯝࠊKA31a ᰴ࡜ẚ㍑ࡋ࡚ a924E1 ᰴࡢ ᅽຊឤཷᛶࡀ☜ㄆ࡛ࡁࡓࠋ஧ಸయᰴࡣࠊKA31a ᰴ࡜ྠ⛬ᗘࡢᅽຊ⪏ᛶࢆ♧ࡍ⩌ ࡜a924E1 ᰴ࡟༉ᩛࡍࡿᅽຊឤཷᛶࢆ♧ࡍ⩌࡟ศ࠿ࢀࡓࠋ ➨5 㡯 㔝⏕ᆺ࣑ࢺࢥࣥࢻࣜ࢔࡟ࡼࡿḞኻ㑇ఏᏊࡢ⿵᏶  ࿧྾Ḟᦆ㓝ẕᰴ࡜㔝⏕ᆺ㓝ẕᰴࡢ᥋ྜ࡟࠾࠸࡚ࠊ࿧྾Ḟᦆᆺ࣑ࢺࢥࣥࢻࣜ࢔ ࡣ㔝⏕ᆺ࣑ࢺࢥࣥࢻࣜ࢔࡟୍ᐃࡢ๭ྜ࡛⨨᥮ࡉࢀࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࠋࡑ ࡇ࡛ࠊa924E1 ᰴࡢ࿧྾Ḟᦆ࠾ࡼࡧᅽຊឤཷᛶࡢ⾲⌧ᆺࡀసฟࡋࡓ஧ಸయᰴ࡬࡝ ࡢࡼ࠺࡟ཷࡅ⥅ࡀࢀࡿࡢ࠿ホ౯ࡋࡓࠋTTC ᰁⰍࡢ⤖ᯝࠊ஧ಸయᰴࡣ TTC ࣏ࢪࢸ ࢕ࣈ(࿧྾ᶵ⬟᭷)࠾ࡼࡧ TTC ࢿ࢞ࢸ࢕ࣈ(࿧྾ᶵ⬟పୗ) ࡢ 2 ࡘࡢ⾲⌧ᙧ㉁ࢆ♧ ࡍ⩌࡟ศ㞳ࡋࡓ(ᅗ 1)ࠋTTC ࣏ࢪࢸ࢕ࣈ⩌ࡣ KA31a ᰴ࡜ྠ⛬ᗘࡢᅽຊ⪏ᛶࢆ♧ ࡍ⩌࡜ྠࡌ࡛࠶ࡗࡓࠋྠᵝ࡟TTC ࢿ࢞ࢸ࢕ࣈ⩌ࡣ a924E1 ᰴ࡟༉ᩛࡍࡿᅽຊឤ

43

ཷᛶࢆ♧ࡋࡓ⩌࡜ྠࡌ࡛࠶ࡗࡓࠋࡲࡓࠊTTC ࣏ࢪࢸ࢕ࣈ⩌᳨࡛ฟࡉࢀࡿCOX1 㑇ఏᏊࡀࠊTTC ࢿ࢞ࢸ࢕ࣈ⩌࡛ࡣḞኻࡋ࡚࠸ࡿࡇ࡜ࢆ PCR ࡟ࡼࡾ☜ㄆࡋࡓ(ᅗ 5)ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊᅽຊឤཷᛶ⬟ࡀ࣓ࣥࢹࣝᘧ࡟㑇ఏࡍࡿࡢ࡛ࡣ࡞ࡃࠊ࿧྾Ḟ ᦆ࣑ࢺࢥࣥࢻࣜ࢔࡜ඹ࡟⣽⬊㉁㑇ఏࡋࡓࡇ࡜ࢆ♧၀ࡍࡿࠋ

44 ➨ ➨4 ⠇ ⪃ᐹ ᮏ❶࡛ࡣࠊDNA ࣐࢖ࢡࣟ࢔ࣞ࢖ゎᯒ࡛♧၀ࡉࢀࡓ࣑ࢺࢥࣥࢻࣜ࢔࡜ᅽຊឤཷ ᛶࡢ㛵㐃ᛶ࡟ࡘ࠸࡚↔Ⅼࢆᙜ࡚࡚ゎᯒࡋࡓࠋTTC ᰁⰍ࡟ࡼࡾ࣑ࢺࢥࣥࢻࣜ࢔ࡢ ࿧྾ᶵ⬟ࢆホ౯ࡋࡓ࡜ࡇࢁࠊa924E1 ᰴࡢ࿧྾ᶵ⬟ࡀపୗࡋ࡚࠸ࡿࡇ࡜ࡀ♧ࡉࢀ ࡓ(ᅗ 1)ࠋ࣑ࢺࢥࣥࢻࣜ࢔ DNA ࢆᑐ㇟࡜ࡋࡓ PCR ࡟ࡼࡗ࡚COX1 㑇ఏᏊࢆࢥ ࣮ࢻࡍࡿ㡿ᇦࡢḞኻࡀ♧ࡉࢀࡓ(ᅗ 5)ࠋCOX1 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ẕᰴࡣ඲࡚ࡢ࣑ࢺࢥࣥࢻࣜ࢔DNA ࢆḞᦆ ࡋ࡚࠸ࡿࡀࠊa924E1 ᰴࡣ COX1 㑇ఏᏊࡢࡳࢆḞኻࡋࡓ㠀ᖖ࡟⛥࡞࿧྾Ḟᦆᰴ ࡛࠶ࡾࠊ࣑ࢺࢥࣥࢻࣜ࢔࡜࿧྾ᶵ⬟࡟㛵ࡍࡿ◊✲࡟࠾࠸࡚ࡶ᭷┈࡞ኚ␗ᰴ࡛࠶ ࡿࠋ  సฟࡋࡓ஧ಸయᰴ࡟࠾࠸࡚ࠊCOX1 㑇ఏᏊࡢḞኻࡣࠊ࣑ࢺࢥࣥࢻࣜ࢔࡜ඹ࡟ ⣽⬊㉁㑇ఏࡋࡓ(ᅗ 1)ࠋCOX1 㑇ఏᏊࢆḞኻࡋࡓ࣑ࢺࢥࣥࢻࣜ࢔ࢆᣢࡘ࿧྾ᶵ⬟ ࡀపୗࡋࡓ஧ಸయᰴࡣࠊCOX1 㑇ఏᏊḞኻ࣑ࢺࢥࣥࢻࣜ࢔ࡀ㔝⏕ᆺ࣑ࢺࢥࣥࢻ ࣜ࢔࡟⨨ࡁ᥮࠼ࡽࢀࡓ࿧྾ᶵ⬟ࢆ⥔ᣢࡋࡓ஧ಸయᰴ࡜ẚ㍑ࡋ࡚ࠊ᭷ព࡟㧗࠸ᅽ ຊឤཷᛶࢆ♧ࡋࠊࡑࡢᅽຊឤཷᛶ⬟ࡣa924E1 ᰴ࡟༉ᩛࡍࡿࡇ࡜ࡀ♧ࡉࢀࡓ(ᅗ

45 4)ࠋࡇࢀࡽࡢ⤖ᯝࡣࠊCOX1 㑇ఏᏊࡢḞኻࡀ a924E1 ᰴ࡟ᅽຊឤཷᛶ⬟ࢆ௜୚ࡋ ࡚࠸ࡿ୍ᅉ࡛࠶ࡿࡇ࡜ࢆ♧ࡋ࡚࠸ࡿࠋ࣑ࢺࢥࣥࢻࣜ࢔ᶵ⬟ࡀᅽຊឤཷᛶ⬟࡟ᙉ ࡃ㛵㐃ࡍࡿ࡜࠸࠺ࡇࢀࡽࡢ⤖ᯝࡣ๓౛ࡀ↓࠸ࡀࠊᇶ♏ⓗ࡞ᅽຊឤཷᛶᶵᵓࢆ⌮ ゎࡍࡿࡓࡵࡢࣈࣞ࢖ࢡࢫ࣮ࣝ࡟࡞ࡿྍ⬟ᛶࡀ࠶ࡿࠋ  ࣑ࢺࢥࣥࢻࣜ࢔ࡣࠊATP ࡸ NADHࠊᵝࠎ࡞࢔࣑ࣀ㓟ࡢ୰㛫⏘≀➼ࢆ⏕ᡂࡍࡿ ୺せ࡞௦ㅰ⤒㊰ࡢ୰ᚰⓗ࡞ᙺ๭ࢆᢸ࠺ࠋ࠸ࡃࡘ࠿ࡢ໬ྜ≀ࡣቑṪ࡟ᚲ㡲࡛ࡣ࡞ ࠸ࡀࠊ࣑ࢺࢥࣥࢻࣜ࢔ᶵ⬟ࡢపୗ࡟ࡼࡗ࡚୺せ࡞௦ㅰࡀ೵Ṇࡍࡿࡇ࡜ࡣࠊከࡃࡢ ࢫࢺࣞࢫ⪏ᛶࢆ㜼ᐖࡍࡿࡇ࡜࡟ࡘ࡞ࡀࡿࠋ౛࠼ࡤࠊࣉࣟࣜࣥࠊ࢔ࣝࢠࢽࣥࠊࢢࣝ ࢱ࣑ࣥ㓟ࠊᒀ⣲➼ࡢ௦ㅰ࡟㛵㐃ࡍࡿ㓝⣲ࡣ࣑ࢺࢥࣥࢻࣜ࢔࡟ᒁᅾࡋ࡚࠾ࡾࠊࡇࢀ ࡽࡣ㓟໬ࢫࢺࣞࢫࠊ㧗 ࢫࢺࣞࢫࠊ෾⤖⼥ゎࢫࢺࣞࢫ➼ࡢࢫࢺࣞࢫ⪏ᛶ࡟ᐤ୚ࡋ ࡚࠸ࡿࠋࡇࢀࡽࡢ⤖ᯝࡣࠊa924E1 ᰴ࡟ᅽຊឤཷᛶ⬟ࢆ௜୚ࡋࡓせᅉ࡜ࡋ࡚ࠊ COX1 㑇ఏᏊࡢḞኻ࡟ࡼࡿ࣑ࢺࢥࣥࢻࣜ࢔ࡢᶵ⬟୙඲࡟㉳ᅉࡍࡿ┤᥋ⓗ࡞ᙳ㡪 ࡛ࡣ࡞ࡃࠊ࣑ࢺࢥࣥࢻࣜ࢔ࡢᶵ⬟୙඲࡟క࠺ࢫࢺࣞࢫ⪏ᛶ࡟ᐤ୚ࡍࡿ௦ㅰ⏘≀ ࡢῶᑡ࡟ࡼࡗ࡚ᵝࠎ࡞ࢫࢺࣞࢫ࡟ᑐࡍࡿ⪏ᛶࡀపୗࡋࡓྍ⬟ᛶࡀ⪃࠼ࡽࢀࡿࠋ  ୍⯡ⓗ࡟࿧྾Ḟᦆ㓝ẕᰴࡣ඲࡚ࡢ࣑ࢺࢥࣥࢻࣜ࢔DNA ࢆḞᦆࡋ࡚࠾ࡾࠊࡑࡢ ࢚ࢱࣀ࣮ࣝⓎ㓝⬟࠾ࡼࡧቑṪ⬟ࡣ㔝⏕ᆺᰴ࡜ẚ㍑ࡋ࡚኱ࡁࡃῶᑡࡍࡿࠋࡋ࠿ࡋ ࡞ࡀࡽࠊᅽຊឤཷᛶኚ␗ᰴa924E1 ᰴࡣCOX1 㑇ఏᏊḞኻ࡟㉳ᅉࡍࡿ࣑ࢺࢥࣥ ࢻࣜ࢔ᶵ⬟୙඲ࢆᘬࡁ㉳ࡇࡋ࡚࠸ࡿ࡟ࡶ㛵ࢃࡽࡎࠊKA31a ᰴ࡜ྠ➼ࡢ࢚ࢱࣀ࣮

46

ࣝⓎ㓝⬟(Shigematsu et al., 2010a)࠾ࡼࡧቑṪ⬟(ᅗ 3)ࢆ᭷ࡍࡿࠋBrauer ࡽ (2005)࡟ࡼࡗ࡚ࠊࢢࣝࢥ࣮ࢫ⃰ᗘࡀ 0.5%௨ୖࡢ᮲௳࡛ᇵ㣴ࡍࡿሙྜࠊS. cerevisiae ࡢቑṪࡣ୺࡟࢚ࢱࣀ࣮ࣝⓎ㓝࡟౫Ꮡࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿࡇ࡜ ࠿ࡽࠊa924E1 ᰴ࠾ࡼࡧ KA31a ᰴࡣ࢚ࢱࣀ࣮ࣝⓎ㓝ࡍࡿࡇ࡜࡟ࡼࡾぢ࠿ࡅୖྠ ➼ࡢቑṪ⬟ࢆ᭷ࡋ࡚࠸ࡓ࡜⪃࠼ࡽࢀࡿࠋࡲࡓࠊࡇࢀࡽࡢ⤖ᯝࡣࠊCOX1 㑇ఏᏊࡢ Ḟኻ࡟㉳ᅉࡍࡿ௦ㅰࡢኚ໬ࡀ࢚ࢱࣀ࣮ࣝⓎ㓝⬟࡟ᙳ㡪ࢆཬࡰࡉࡎ࡟ᅽຊឤཷᛶ ⬟ࢆ௜୚ࡋ࡚࠸ࡿࡇ࡜ࢆ♧၀ࡋ࡚࠸ࡿࡇ࡜ࡣࠊPReF ᢏ⾡ࡢᐇ⏝໬࡟ྥࡅ࡚᭷┈ ࡛࠶ࡿࠋ  ᮏ❶ࡢ⤖ᯝࢆࡲ࡜ࡵࡿ࡜ࠊa924E1 ᰴࡢᅽຊឤཷᛶ⬟ࡣ࣑ࢺࢥࣥࢻࣜ࢔ࡢᶵ⬟ ୙඲࡟ࡼࡾ௜୚ࡉࢀࡓ࡜⪃࠼ࡽࢀࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊᅽຊឤཷᛶࢆ௜୚ࡍࡿᶵᵓ ࡢ┤᥋ⓗ࡞ドᣐࡣ᫂ࡽ࠿࡜࡞ࡽ࡞࠿ࡗࡓࡀࠊa924E1 ᰴࡢ࣑ࢺࢥࣥࢻࣜ࢔ࢆ௚ࡢ 㓝ẕᰴࡢ࣑ࢺࢥࣥࢻࣜ࢔࡟⿵᏶ࡍࡿࡇ࡜࡛ᐜ᫆࡟⏘ᴗ⏝㓝ẕᰴ࡟ᅽຊឤཷᛶ⬟ ࢆ௜୚ࡍࡿࡇ࡜ࡀᮇᚅ࡛ࡁࡿࠋ

47

➨

➨

4 ❶ ࣓ࢱ࣑࣎ࣟࢡࢫ࡟ࡼࡿᅽຊឤཷᛶᶵᵓࡢゎᯒ



 ➨1 ⠇ ⥴ゝ ➨1 㡯 ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒ  ࣓ࢱ࣑࣎ࣟࢡࢫࡣࠊ᭷ᶵ໬ྜ≀ࠊ࢔࣑ࣀ㓟ࠊ᰾㓟➼ࡢ⣽⬊ෆ௦ㅰ⏘≀ࢆࢱ࣮ࢤ ࢵࢺ࡜ࡋࡓ⥙⨶ⓗゎᯒᢏ⾡࡛࠶ࡿ(⚟ᓮ, 2013)ࠋ࣓ࢱ࣑࣎ࣟࢡࢫࡢ኱ࡁ࡞≉ᛶࡣ ࡑࡢ୍⯡ᛶ࡛࠶ࡿࠋDNA ࡸ RNA ࢆ⥙⨶ⓗ࡟ゎᯒࡍࡿࢤࣀ࣑ࢡࢫࠊࢱࣥࣃࢡ㉁ ࢆ⥙⨶ⓗ࡟ゎᯒࡍࡿࣉࣟࢸ࣑࢜ࢡࢫ➼࡟ࡼࡿゎᯒ࡛ࡣࠊࡑࡢゎᯒࢧࣥࣉࣝ࡟≉ ᭷ࡢሷᇶ㓄ิ➼ࡢ᝟ሗࡀᚲせ࡛࠶ࡿࠋࡑࡢ୍᪉ࠊ࣓ࢱ࣑࣎ࣟࢡࢫࡢゎᯒᑐ㇟ࡣ௦ ㅰ⏘≀࡛࠶ࡾࠊᙜ↛ࡢࡇ࡜࡞ࡀࡽࡑࢀࡽࡣࣄࢺ࡛ࡶ኱⭠⳦࡛ࡶ⏕≀㛫࡛஫᥮ᛶ ࢆ᭷ࡍࡿ໬ྜ≀࡛࠶ࡿሙྜࡀከ࠸ࠋࡋ࠿ࡋࠊ࣓ࢱ࣑࣎ࣟࢡࢫ࡛ゎᯒ࡛ࡁࡿ௦ㅰ⏘ ≀࡟ᶵ⬟ᛶࡀ࠶ࡿ࡜ࡣ㝈ࡽ࡞࠸ࡓࡵ࡟ࠊ⏕≀Ꮫⓗ࡞⌮⏤௜ࡅࡀ㞴ࡋ࠸࡜࠸࠺ၥ 㢟ࡶ࠶ࡿࠋ౛࠼ࡤࠊ࠶ࡿ௦ㅰ⏘≀ࡀቑຍࡋࡓሙྜࠊᚲせ࡟࡞ࡗࡓࡓࡵ࡟⵳✚ࡋࡓ ࡢ࠿ࠊ୙せ࡟࡞ࡗࡓࡢ࠿ࡽ⵳✚ࡋࡓࡢ࠿ࡢゎ㔘ࡣࠊูࡢᐇ㦂⤖ᯝ࡛⌮⏤௜ࡅࢆ⾜ ࠺ᚲせࡀ࠶ࡿ(㔝ᮧ, ᒾᶫ, 2013)ࠋ

48 ➨2 㡯 ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒࡢᛂ⏝౛ ከ✀ከᵝ࡞໬ྜ≀ࢆศᯒࡍࡿ࣓ࢱ࣑࣎ࣟࢡࢫ࡟᭱ࡶࡼࡃ⏝࠸ࡽࢀࡿᢏ⾡ࡣࠊ ㉁㔞ศᯒ࡛࠶ࡿࠋゎീᗘ࡜෌⌧ᛶ࡟ඃࢀࡓ࢞ࢫࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮㉁㔞ศᯒ (GC/MS)ࡸᾮయࢡ࣐ࣟࢺࢢࣛࣇ࢕࣮㉁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ᯒࡍࡿࡓࡵ࡟࢟ࣕࣆࣛ ࣮ࣜ㟁ẼὋື㉁㔞ศᯒἲ(CE/MS)ࢆ⏝࠸ࡓ⣽⬊ෆ௦ㅰ⏘≀ࡢ⥙⨶ⓗゎᯒࢆ⾜࠸ࠊ ࢫࢺࣞࢫᛂ⟅ゎᯒ࡟࠾ࡅࡿ࣓ࢱ࣑࣎ࣟࢡࢫࡢ᭷⏝ᛶࢆ♧ࡋࡓࠋࡲࡓࠊࢤࣀ࣑ࢡࢫ ࡜࣓ࢱ࣑࣎ࣟࢡࢫࢆ⤌ࡳྜࢃࡏࡿࡇ࡜࡛ࠊᶵ⬟᝟ሗ࡜௦ㅰ᝟ሗࢆྠ᫬࡟ゎᯒࡍ ࡿࡇ࡜ࡶሗ࿌ࡉࢀ࡚࠸ࡿ(⏣୰ࡽ, 2013)ࠋᶵ⬟᝟ሗࡣཎᅉ࡛࠶ࡾࠊ௦ㅰ᝟ሗࡣ⤖ ᯝ࡛࠶ࡿࠋࡇࢀࡽࢆ⤌ࡳྜࢃࡏࡿࡇ࡜࡛ࠊ࡝ࡢࡼ࠺࡞㑇ఏᏊࡢⓎ⌧ࡢኚ໬ࡀཎᅉ ࡜࡞ࡗ࡚ࠊࢫࢺࣞࢫ➼࡟㐺ᛂࡍࡿ⤖ᯝ࡜࡞ࡿࡢ࠿ࠊࡼࡾ῝ࡃ⪃ᐹࡍࡿࡇ࡜ࡀ࡛ࡁ ࡿࠋ ➨3 㡯 ࢔ࣝࢠࢽࣥ ࢔ࣝࢠࢽࣥࡣࢢ࢔ࢽࢪࣥ࡜㢮ఝࡋࡓ࢔࣑ࣀ㓟ഃ㙐ࢆ᭷ࡍࡿሷᇶᛶ࢔࣑ࣀ㓟ࡢ

49

ࡍࡿኚᛶ๣࡛࠶ࡿࡓࡵࠊࢢ࢔ࢽࢪࣥ࡟㢮ఝࡋࡓഃ㙐ࢆᣢࡘ࢔ࣝࢠࢽࣥࡶจ㞟ࡋ

ࡓࢱࣥࣃࢡ㉁ࡢ࣮ࣜ࣍ࣝࢹ࢕ࣥࢢࢆ⿵ຓࡍࡿ᭷ᮃ࡞ῧຍ≀࡛࠶ࡿ(Tsumoto et al., 2004; Lyutoya et al., 2007)ࠋࡑࢀᨾ࡟ࠊ࢔ࣝࢠࢽࣥࡣ㓝ẕ࡟࠾࠸࡚㓟໬ࡸ෾ ⤖࡟ᑐࡍࡿࢫࢺࣞࢫ⪏ᛶ࡟㛵୚ࡍࡿࡇ࡜ࡀሗ࿌ࡉࢀ࡚࠸ࡿ(Morita et al., 2002; Nishimura et al., 2010)ࠋ㧗ᅽࡣ㠀ඹ᭷⤖ྜࡢ୙Ᏻᐃ໬ࢆᘬࡁ㉳ࡇࡋ࡚ࢱࣥࣃࢡ ㉁ࡢኚᛶࢆಁ㐍ࡍࡿࠋࡋ࠿ࡋ࡞ࡀࡽࠊ࢔ࣝࢠࢽࣥࡢ㧗ᅽࢫࢺࣞࢫ࡟ᑐࡍࡿຠᯝࡣ ᮍࡔ࠶ࡁࡽ࠿࡜࡞ࡗ࡚࠸࡞࠸ࠋ  ➨4 㡯 ᐇ㦂┠ⓗ ➨2 ❶࠾ࡼࡧ➨ 3 ❶ࡢ⤖ᯝ࠿ࡽࠊa924E1 ᰴࡢᅽຊឤཷᛶ⬟ࡀCOX1 㑇ఏᏊ ࡢḞኻ࡟㉳ᅉࡍࡿ࣑ࢺࢥࣥࢻࣜ࢔ࡢᶵ⬟୙඲࡟ࡼࡾ௜୚ࡉࢀࡓࡇ࡜ࡀ♧၀ࡉࢀ ࡓࠋᮏ㡯࡛ࡣࠊ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒ࡟ࡼࡾࠊ࣑ࢺࢥࣥࢻࣜ࢔ࡢᶵ⬟୙඲࡟ࡼࡾᅽ ຊឤཷᛶࢆᘬࡁ㉳ࡇࡍ࣓࢝ࢽࢬ࣒ࢆゎᯒࡋࡓࠋࡲࡓࠊ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒ࡟ࡼࡾ ᵝࠎ࡞ࢫࢺࣞࢫ⪏ᛶ࡟ᐤ୚ࡍࡿ࢔ࣝࢠࢽࣥࡢῶᑡࡀ♧ࡉࢀࡓࡓࡵ࡟ࠊ࢔ࣝࢠࢽ ࣥࡢᅽຊ⪏ᛶ࡬ࡢᐤ୚࡟ࡘ࠸࡚ࡶేࡏ࡚ゎᯒࡋࡓࠋ

50 ➨ ➨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 ⥺↷ᑕ࡟ࡼࡿ✺↛ኚ␗࡟ࡼࡾྲྀᚓࡋࡓ(Shigematsu et al., 2010a)ࠋࡇࢀࡽࡢ㓝ẕᰴ ࡣࠊ᪂₲⸆⛉኱Ꮫ 㔜ᯇ ஽ ᩍᤵࡼࡾศ୚ࡋ࡚࠸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᭤⥺ࡣHasegawa ࡽ(2012)ࡢ᪉ἲ࡟ᚑ࠸ࠊ Bio Microplate Reader HiTSTM _{(Scinics, Ⲉᇛ, ᪥ᮏ)ࢆ⏝࠸࡚ 30°Cࠊ120 h ࡢ᮲௳࡛}

 ᐃࡋࡓࠋ

➨3 㡯 ࣓ࢱ࣑࣎ࣟࢡࢫゎᯒ᪉ἲ

௦ㅰ⏘≀ࡢᢳฟἲࡣHuman Metabolomics Technologies (HMT; ᒣᙧ, ᪥ᮏ)ࡢࣉ ࣟࢺࢥࣝ࡟ᚑࡗࡓࠋCE/MS ࡟ࡼࡿ௦ㅰ⏘≀ࡢ᳨ฟࡣ HMT ࡟ጤクࡋࡓࠋa924E1