ᚑくᄢቇℂᎿቇ⎇ⓥႎ๔
J. Fac. Sci. Tech., Seikei Univ.
Vol.46 No.2 (2009) pp.31-32㧔⎇ⓥㅦႎ㧕㩷㩷㔚ሶવ㆐♽ㆮવሶ 㪪㪛㪟㪚 ߩᣂⷙ㫍㪸㫉㫀㪸㫅㫋 ⊒⷗ߣᯏ⢻⸃ᨆ㩷૒⮮ ⩿‫ ⼱⩲ ޘ‬㤗Ꮧ ਭን ኼ 㩷

Identification of alternative splicing transcripts for the SDHC geneand distribution of their mRNA in human tissues
Nana SATOH*1,Maki SUGAYA*2,Hisashi HISATOMI*3(Received September 18, 2009)㧝㧚⢛ ᥊⊒⃻㊂ࠍ RT real-time PCR ߢ⺞ᩏߒߚߩߢႎ๔ߔࠆ‫ޕ‬ ࡅ࠻ߩ૕ߪ⚂ 60 ళ୘ߩ⚦⢩ߢ᭴ᚑߐࠇߡ߅ࠅ㧘1 ߟߩ㧞㧚᧚ᢱߣᣇᴺ⚦⢩ߩਛߦࡒ࠻ࠦࡦ࠼࡝ࠕߪᢙ⊖୘ሽ࿷ߒߡ޿ࠆ‫࠻ࡒޕ‬ࠦࡦ࠼࡝ࠕߩਥⷐߥᓎഀߣߒߡߪ㧘㘩੐߆ࠄ៨ขߒߚ὇ ࡅ࠻ᄢ⣺≸⚦⢩ᩣ DLD-1 ⚦⢩߆ࠄ㧘GuickGene RNA᳓ൻ‛㧘⢽⢌㧘࠲ࡦࡄࠢ⾰ߥߤߩᩕ㙃⚛ߩ ATP ߳ߩᄌ឵cultured cell HC kit S (ን჻ࡈࠖ࡞ࡓ)ࠍ↪޿ߡ total RNAࠍ᛽಴ߒ㧘Random primer (GIBCO)ߦࠃࠅ㧘cDNA ࠍวᚑߣ⚦⢩߳ߩଏ⛎ߢ޽ࠆ‫ߪࠕ࡝࠼ࡦࠦ࠻ࡒߦ߁ࠃߩߎޕ‬㧘ߒߚ‫ޕ‬SDHC ߩ exon1 ߦ Forward primer ࠍ exon6 ߦ Reverse⚦⢩ߩ᭽‫ߥޘ‬ᵴേߦᔅⷐߥࠛࡀ࡞ࠡ࡯ߩᄢඨࠍวᚑߒߡprimer ࠍ⸳ቯߒ㧘วᚑߒߚ cDNA ࠍ template ߣߒߡ޿ࠆ㊀ⷐߥ⚦⢩ዊེቭߢ޽ࠅ㧘ߤߩ⚦⢩ߦ߽᥉ㆉ⊛ߦሽ

RT-PCR ᴺࠍⴕߞߚ‫ޕ‬RT-PCR ᴺߦࠃࠅᓧࠄࠇߚ PCR Ⴧ࿷ߔࠆ‫ޕ‬ᚒ‫ߪޘ‬㧘ߎߩࡒ࠻ࠦࡦ࠼࡝ࠕౝ⤑ߦዪ࿷ߔࠆ
succinate dehydrogenase (SDH) ߦ⌕⋡ߒߚ‫ޕ‬SDH ߪࠦࡂ᏷↥‛ࠍ♖⵾ᓟ㧘
Applied Biosystems 3130 Genetic Analyzer

cycle ߩ৻┵ࠍᜂ޿㧘๭ๆ㎮ⶄว૕Τߣߒߡᵴേߒߡ޿ࠆ‫ޕ‬ASV ߪ㧘޿ߕࠇ߽ 5’߅ࠃ߮ 3’ RACE ᴺߦࠃࠅᰳᄬ㗔ၞ৻ᣇ㧘ࠦࡂࠢ㉄ࠍࡈࡑ࡞㉄߳㉄ൻㆶరߒߚ㓙ߦ↢ߓߚ㔚એᄖߩႮၮ㈩೉ߩ଻ሽࠍ⏕⹺ߒߚ‫ߚ߹ޕ‬㧘㧣⒳㘃ߩࡅ࠻ሶࠍᰴߩⶄว૕߳વ㆐ߔࠆ㔚ሶવ㆐♽㉂⚛ߣߒߡ߽௛޿↱᧪ฦ⤳ེߦ߅ߌࠆቢో㐳 SDHC mRNA ߅ࠃ߮ฦ ASV1)

ࠢ㉄ࠍࡈࡑ࡞㉄߳㉄ൻㆶరߔࠆߎߣߢ tricarboxylic acidߦߡႮၮ㈩೉ࠍ⸃ᨆߒ㧘ASV ࠍหቯߒߚ‫ޕ‬หቯߐࠇߚmRNA ㊂ߩ᷹ቯࠍ RT real-time PCR ᴺߦࠃࠅⴕߞߚߡ޿ࠆߚ߼㧘㔚ሶવ㆐♽ⶄว૕Τߣ߽๭߫ࠇߡ޿ࠆ‫ߎޕ‬ߩ SDH ߪ 4 ߟߩ࠲ࡦࡄࠢ⾰߆ࠄ᭴ᚑߐࠇߡ޿ࠆⶄว૕ߢ(iCycler iQ real-time Detection System㧘BIO-RAD)‫ޕ‬6-FAM޽ࠅ㧘ߎߩ߁ߜߩ৻ߟ߇ Succinate dehydrogenase subunitߢⰯశᮡ⼂ߒߚ TaqMan probe ߪ exon4 ߦ⸳ቯߒ㧘ቢో㐳C (SDHC)ߢ޽ࠆ‫ޕ‬SDHC ߪ ubiquinone ㉄ൻㆶర㉂⚛ߣߒmRNA ߅ࠃ߮ฦ ASV ߦ౒ㅢߥ probe ߣߒߚ‫ޕ‬ߡ௛ߊߎߣߢ㔚ሶߩᯅᷰߒࠍߒ㧘SDH ߩᜬߟ 2 ߟߩ㉂⚛

ᯏ⢻ࠍ⋥ធ⚿߮ߟߌࠆᓎഀࠍᨐߚߒߡ޿ࠆ‫ޕ‬㧟㧚⚿ ᨐ SDHC ㆮવሶߪ 1q21 ߦ૏⟎ߒ㧘49608 bp ߩ DNA ਛߦ6 ߟߩ exon ࠍᜬߟ‫ޕ‬Open Reading Frame ߆ࠄߪ 507 Ⴎၮ㧘 HCT-15 ⚦⢩↱᧪ cDNA ࠍ↪޿ߡ SDHC mRNA ࠍჇ᏷ߩ⊒⃻⺞ᩏߩㆊ⒟ߢ㧘exon 3 ߅ࠃ߮ exon 5 ߇ᰳᄬߔࠆ 2᣿ࠄ߆ߦ⍴޿ 2 ⒳㘃ߩჇ᏷↥‛ (464 bp ߅ࠃ߮ 402 bp)߇ᚒ‫ ࠄࠇߎߚߒ⷗⊒߇ޘ‬ASV ߪ㧘paraganglioma ࠍߪߓ߼ߣ㈩೉ࠍ࠳ࠗ࡟ࠢ࠻ࠪ࡯ࠢࠛࡦࠬᴺߦࠃࠅ᳿ቯߒߚ⚿ᨐ㧘

169 ࠕࡒࡁ㉄߇⠡⸶ߐࠇࠆ‫ޕ‬ᚒ‫ߪޘ‬㧘ࡅ࠻ SDHC ㆮવሶߒߚ⚿ᨐ㧘ቢో㐳 mRNA ↱᧪Ⴧ᏷↥‛ (566 bp) ࠃࠅ߽⒳㘃ߩᣂⷙ alternative splicing variant (ASV) ࠍ⊒⷗ߒߚ‫ޕ‬ߔࠆ᭽‫∔ߥޘ‬ᖚߢ⏕⹺ߐࠇߪߓ߼ߡ޿ࠆ

1,2)‫⎇ᧄޕ‬ⓥߢߪ㧘

SDHC ASV ߩᯏ⢻⸃ᨆࠍⴕ߁ߚ߼㧘ߎࠇࠄ ASV ߩ⤳ེ೎⏕⹺ߐࠇߚ (Figure 1A)‫ߦࠄߐޕ‬㧘ߎࠇࠄჇ᏷↥‛ߩႮၮቢో㐳 mRNA ↱᧪Ⴧ᏷↥‛ (566 bp) ߪ࠺࡯࠲ࡌ࡯ࠬ਄

ߦ߅ߌࠆ SDHC mRNA ߩႮၮ㈩೉ߣቢోߦ৻⥌ߒߚߚ߼ߦ㧘Full length isoform ߣ᳿ቯߒߚ‫ߚ߹ޕ‬ห᭽ߩᣇᴺߢ 2*1⒳㘃ߩ⍴޿Ⴧ᏷↥‛ࠍ⸃ᨆߒߚ⚿ᨐ㧘464 bp ߩჇ᏷↥‛㧦ℂᎿቇ⎇ⓥ⑼ℂᎿቇኾ᡹ඳ჻೨ᦼ⺖⒟*2㧦‛⾰↢๮ℂᎿቇ⑼ഥᢎ
*3
㧦‛⾰↢๮ℂᎿቇ⑼ಎᢎ᝼(hisatomi@st.seikei.ac.jp)

ߪ Full length isoform ߆ࠄ exon3 ߇ᰳᄬߔࠆ isoform (delta㧙㧙೙ߔࠆߣផ᷹ߐࠇࠆ‫ߚ߹ޕ‬㧘delta5 isoform ߦ߅޿ߡߪ㧘3) ߅ࠃ߮ 402 bp ߩჇ᏷↥‛ߪ Full length isoform ߆ࠄexon5 ߇ᰳᄬߔࠆ delta5 ߣหቯߒߚ‫߅ߥޕ‬㧘delta5 ߢߪ

dominant negative inhibitor ߹ߚߪ SDHC ߣߪ⇣ߥࠆ࠲ࡦframe shift ߇↢ߓ㧘stop codon ߇ 3’ non coding region ߳⒖

ࡄࠢ⾰ߣߒߡᯏ⢻ߔࠆߣ޿߁ਔᣇะ߆ࠄߩน⢻ᕈ߇ផ᷹ߐࠇࠆ‫ߒ߆ߒޕ‬㧘SDHC ASV ࠍࠢࡠ࡯࠾ࡦࠣߒߚ plasmidⴕߒߚߎߣߦࠃࠅ㧘70 ࠕࡒࡁ㉄߇ᣂߚߦ⊒↢ߒߚ‫ࠇߎޕ‬ࠄ ASV ߩ⚦⢩ౝ⊒⃻㊂ࠍ㧣⒳㘃ߩࡅ࠻↱᧪ฦ⤳ེ೎ߦࠍ↪޿ߡㆮવሶዉ౉ߒ㧘ㆊ೾⊒⃻ߐߖߚߘࠇߙࠇߩ⚦⢩RT real-time PCR ᴺߢ᷹ቯߒߚ⚿ᨐ㧘ߎࠇࠄ ASV ߩฦ⤳ߢ SDHC Full length mRNA ㊂ࠍ RT-PCR ᴺߦࠃࠅቯ㊂ߒེߦ߅ߌࠆሽ࿷Ყߪ᥉ㆉ⊛ߢ޽ࠆߎߣ߇᣿ࠄ߆ߣߥߞߚߚ⚿ᨐ㧘ࠪࠣ࠽࡞ᒝᐲߩᏅ⇣ߪⷰኤߐࠇߕ㧘⊒⃻㊂ߦᄌ(Figure 1B)‫ޕ‬ൻߪߥ߆ߞߚ‫⚿ߩߎޕ‬ᨐࠃࠅ㧘SDHC ASV ߪㆊ೾⊒⃻ߒߡ߽㧘dominant negative inhibitor ߣߒߡᯏ⢻ߒߡ޿ߥ޿ߎߣ߇␜ໂߐࠇߚ‫ ߚ߹ޕ‬delta5 isoform ߪ㧘⚦⢩⾰ߦὐ࿷ߔࠆࡒ࠻ࠦࡦ࠼࡝ࠕߦዪ࿷ൻߒߡ޿ࠆߎߣ߇⸽᣿ߐࠇߚ‫ޕ‬
ߎࠇߪ㧘Full length isoform ߅ࠃ߮ delta3 isoform ห᭽㧘⚦⢩⾰ߢ࠲ࡦࡄࠢ⾰ߦ⠡⸶ߐࠇߚᓟ㧘ࡒ࠻ࠦࡦ࠼࡝ࠕ߳⒖ⴕߒߡᯏ⢻ߒߡ޿ࠆߣ⠨߃ࠄࠇࠆ‫ߒ߆ߒޕ‬㧘⃻Ბ㓏ߢߪࡒ࠻ࠦࡦ࠼࡝ࠕߦዪ࿷ߒߡ޿ࠆߎߣ߇᣿ࠄ߆ߣߥߞߚߛ
ߌߢ㧘SDHC ASV ߩ૞↪ᯏᐨߪᧂ⸃᣿ߢ޽ࠆ‫ޕ‬ SDHC ߩὐ⓭ὼᄌ⇣ߪ㧘ࡒ࠻ࠦࡦ࠼࡝ࠕ߆ࠄ↥↢ߐࠇ㪝㫀㪾㫌㫉㪼㩷㪈㪅㩷㪜㫏㫇㫉㪼㫊㫊㫀㫆㫅㩷㫇㫉㫆㪽㫀㫃㪼㫊㩷㫆㪽㩷㪪㪛㪟㪚㩷㫊㫇㫃㫀㪺㫀㫅㪾㩷㫍㪸㫉㫀㪸㫅㫋㫊㪅㩷ࠆㆊ೾ߥᵴᕈ㉄⚛⊒↢ࠍ߽ߚࠄߔ‫⚿ߩߘޕ‬ᨐ㧘⠧ൻߩ⃻㪘㪃㩷㪘㫃㫋㪼㫉㫅㪸㫋㫀㫍㪼㩷㫊㫇㫃㫀㪺㫀㫅㪾㩷㫆㪽㩷㪪㪛㪟㪚㩷㫄㪩㪥㪘㪅㩷㪤㪃㩷㫄㫆㫃㪼㪺㫌㫃㪸㫉㩷㫎㪼㫀㪾㪿㫋㩷⽎ߣ⠨߃ࠄࠇࠆ⚦⢩Ⴧᱺ⢻ߩᛥ೙߇↢ߓࠆߎߣ߿㧘↢૕㫄㪸㫉㫂㪼㫉㩷 㪒㩷 㪺㪛㪥㪘㪃㩷 㪺㪛㪥㪘㩷 㫆㪽㩷 㪟㪚㪫㪄㪈㪌㩷 㪺㪼㫃㫃㩷 㫃㫀㫅㪼㪒㩷 㪛㪥㪘㪃㩷 㪾㪼㫅㫆㫄㫀㪺㩷㒐ᓮᯏ᭴ߣߒߡࠕࡐ࠻࡯ࠪࠬࠍ⺃ዉߔࠆࠪࠣ࠽࡞વ㆐⚻㪛㪥㪘㪅㩷㪫㫎㫆㩷㫊㪿㫆㫉㫋㪼㫉㩷㫊㫀㪾㫅㪸㫃㫊㩷㪸㫉㪼㩷㫆㪹㫊㪼㫉㫍㪼㪻㩷㫀㫅㩷㫋㪿㪼㩷㪟㪚㪫㪄㪈㪌㩷㪺㪼㫃㫃㫊㪅㩷〝߇ᵴᕈൻߔࠆߎߣ߇ႎ๔ߐࠇߡ޿ࠆ 4)‫ߚ߹ޕ‬㧘⚦⢩߇㪙㪃㩷㪨㫌㪸㫅㫋㫀㪽㫀㪺㪸㫋㫀㫆㫅㩷㫆㪽㩷㪪㪛㪟㪚㩷㫄㪩㪥㪘㩷㫀㫅㩷㪿㫌㫄㪸㫅㩷㫅㫆㫉㫄㪸㫃㩷㫋㫀㫊㫊㫌㪼㫊㪅㩷ૐ㉄⚛⁁ᘒߣߥࠆߎߣߢ㧘ⴊ▤ᣂ↢ߥߤ≸ߩㅴዷߦ㑐ਈ㫄㪩㪥㪘㩷㪼㫏㫇㫉㪼㫊㫊㫀㫆㫅㩷㫃㪼㫍㪼㫃㫊㩷㪸㫉㪼㩷㫇㫉㪼㫊㪼㫅㫋㪼㪻㩷㪸㫊㩷㫋㪿㪼㩷㫄㪩㪥㪘㩷㪺㫆㫇㫐㩷ߔࠆㆮવሶߩ⊒⃻ࠍ⺞▵ߔࠆ࿃ሶߣߒߡ૞↪ߒߡ޿ࠆߎߣ߇ႎ๔ߐࠇ㧘≸ߣߩ㑐ㅪ߽␜ໂߐࠇߡ޿ࠆ 6)‫੹ޕ‬ᓟ㧘㫅㫌㫄㪹㪼㫉㩷㫇㪼㫉㩷㫫㪾㩷㫋㫆㫋㪸㫃㩷㪩㪥㪘㪅

SDHC ASV ߩ↢૕ౝ૞↪ᯏᐨࠍ⸃᣿ߒ㧘SDHC ASV ߦࠃ㧠㧚⠨ ኤߞߡ⊒∝ߔࠆ∛᳇ߩࡔࠞ࠾࠭ࡓࠍ᣿ࠄ߆ߣߔࠆ੍ቯߢ޽ࠆ‫ޕ‬ ࠛࡀ࡞ࠡ࡯ઍ⻢߇⋓ࠎߥᔃ⤳㧘⣖㧘㛽ᩰ╭㧘␹⚻ߥߤ ߥ߅㧘 ᧄ⎇ⓥߢ⊒⷗ߒߚ ASV ߪ㧘 National Center forߩ⤳ེߪ㧘ࡒ࠻ࠦࡦ࠼࡝ࠕߩᯏ⢻⇣Ᏹߦࠃࠅ㓚ኂࠍฃߌ

Biotechnology Information ߩ࿖㓙࠺࡯࠲ࡌ࡯ࠬߦ⊓㍳߇߿ߔ޿ߎߣ߇ႎ๔ߐࠇߡ޿ࠆ‫ޕ‬ਛߢ߽㧘㔚ሶવ㆐♽㉂⚛

⸵นߐࠇߚ (AB211234㧘AB211235)‫ޕ‬3)ߩ⇣Ᏹߦ઻߁∝଀߇ᄢඨࠍභ߼ࠆ ‫ࠕ࡝࠼ࡦࠦ࠻ࡒޕ‬DNA ߩ⇣Ᏹߦࠃࠆ complex Σ߅ࠃ߮Φߩᯏ⢻ᰳ៊߇ේෳ⠨ᢥ₂࿃ߣߒߡᒁ߈⿠ߎߐࠇࠆࡒ࠻ࠦࡦ࠼࡝ࠕ∛߇৻⥸⊛ߦᐢߊ⍮ࠄࠇߡ޿ࠆ߇㧘ᦨㄭ㧘㔚ሶવ㆐♽㉂⚛ߢ໑৻ᩭ DNA↱᧪ߢ޽ࠆ complex Τߩ⇣Ᏹߦࠃߞߡ⿠ߎࠆ∔ᖚ߽ᄙߊႎ๔ߐࠇߡ޿ࠆ

1) Cascón A, Pita G, Burnichon N et al. J Clin Endocrinol
Metab 94: 1701-1705, 2009.

‫ߜ߁ߩߘޕ‬㧘paraganglioma ߩᖚ⠪ߪ3,4)

2) Pigny P, Cardot-Bauters C, Do Cao C et al. Eur JSDHB㧘SDHC ߅ࠃ߮ SDHD ߩᄌ⇣ࠍᜬߟߎߣ߇ᦨㄭߩ

Endocrinol 160: 227-231, 2009.

⎇ⓥߦࠃࠅ᣿ࠄ߆ߣߥߞߚ ‫⎇ᧄޕ‬ⓥߢߪ㧘paraganglioma
4)ߩේ࿃ㆮવሶߢ޽ࠆน⢻ᕈ߇㜞޿ SDHC ߆ࠄ㧘exon3 ޽3) Dong LF, Low P, Dyason JC et al. Oncogene 27:ㆮવሶ߆ࠄ 102 Ⴎၮᰳᄬߔࠆ delta3 ߪ in-frame mutation

4) Pasini B, McWhinney SR, Bei T et al. Eur J Hum Genet

5)4324-4335, 2008.

ࠆ޿ߪ exon 5 ߇ᰳᄬߒߚᣂߚߥ ASV ࠍ⊒⷗ߒߚ‫ޕ‬SDHC16: 79-88, 2008.

ߢ޽ࠆߚ߼㧘ᰳᄬ㗔ၞએᄖߩ࠲ࡦࡄࠢ⾰ߪ଻ᜬߐࠇߡ޿ࠆ߇㧘162 Ⴎၮ߇ᰳᄬߔࠆ delta5 isoform ߪ flame shift ߦ5) Niemann S, Müller U, Engelhardt D, Lohse P. Hum Genetߣోߊ⇣ߥߞߡ޿ࠆ‫ߡߞ߇ߚߒޕ‬㧘delta3 isoform ߪ6) Selak MA, Armour SM, MacKenzie ED et al. Cancer Cell

113: 92-94, 2003.

ࠃࠅ exon5 ᰳᄬએ㒠ߩࠕࡒࡁ㉄㈩೉߇ Full length isoform

7: 77-85, 2005.

dominant negative inhibitor ߣߒߡᯏ⢻ߒ㧘SDH ᵴᕈࠍᛥ㧙㧙