ᚑくᄢቇℂᎿቇ⎇ⓥႎ๔
J. Fac. Sci. Tech., Seikei Univ.
Vol.45 No.2 (2008) pp.69-70
㧔․೎⎇ⓥ⾌ߦଥࠆ⺰ᢥ㧕)NWVCVJKQPGU[PVJGVCUGO40# ߦ߅ߌࠆᣂⷙ URNKEKPIXCTKCPV ߩ⊒⷗ౝ↰ 㤗㉿ᕺ 㧘⩲⼱ 㤗Ꮧ 㧘ਭን ኼ Identification of an alternative splicing transcript for the glutathione synthetase genein human hepatocarcinoma cell lineMarie Uchida*1, Maki Sugaya*2, Hisashi HISATOMI*3(Received September 27, 2008)㧝㧚⢛ ᥊㧞㧚᧚ᢱߣᣇᴺ ᚒ‫ࠍ࠻ࡅޘ‬฽߻ᅢ᳇ᕈ↢‛ߪ,㉄⚛߆ࠄࠛࡀ࡞ࠡ࡯ࠍ㧙5RNKEKPIXCTKCPV ߩ⊒⷗ᓧࠆߚ߼,ߘߩㆊ⒟ߢ⊒↢ߔࠆᵴᕈ㉄⚛ߦࠃࠆ↢૕ౝ㜞 ࡅ࠻⢄⤳߇ࠎ↱᧪⚦⢩ᩣ Huh-7 ⚦⢩߆ࠄ QuickGene

RNA cultured cell HC kit S (FUJIFILM)ࠍ↪޿ߡ total RNA

ಽሶߩㆊ㉄ൻ߿ DNA ߩ៊்ߥߤߩ࡝ࠬࠢߣᏱߦ㓞ࠅวࠍ᛽಴ߒ㧘random primer(GIBCO)ߦࠃࠅ cDNA ࠍวᚑߒࠊߖߢ޽ࠆ‫ޕ‬ᵴᕈ㉄⚛ߦࠃࠆ↢૕ౝ㉄ൻࠍᛥ೙ߔࠆߚ߼

ߚ‫ޕ‬วᚑߒߚ cDNA ߦ PCR ṁᶧ (1Paq5000TM Reaction

ߦ,ࡅ࠻ߪ⚦⢩ౝߢ᛫㉄ൻ‛⾰ߢ޽ࠆࠣ࡞࠲࠴ࠝࡦࠍว

Buffer㧘0.25 mM dNTP (TAKARA BIO)㧘 30 pM Forwardᚑߒߡ޿ࠆ‫㉄߇ࠄ⥄ߪࡦࠝ࠴࠲࡞ࠣޕ‬ൻߐࠇࠆߎߣߢ㉄ൻဳࠣ࡞࠲࠴ࠝࡦߣߥࠅ,⚦⢩ౝߩᵴᕈ㉄⚛ࠍ㒰෰ߔࠆ‫ޕ‬৻ᣇߢ,㉄ൻߐࠇߚࠣ࡞࠲࠴ࠝࡦߪࠣ࡞࠲࠴ࠝࡦㆶర㉂⚛ߢㆶరߐࠇ,ౣᐲ᛫㉄ൻ‛⾰ߣߒߡ૶↪ߐࠇࠆ‫ߩߎޕ‬ㆶరဳࠣ࡞࠲࠴ࠝࡦߪࠣ࡞࠲ࡒࡦ㉄,ࠪࠬ࠹ࠗࡦ,ࠣ࡝ࠪࡦߩ㧟ߟߩࠕࡒࡁ㉄߆ࠄᚑࠆ࠻࡝ࡍࡊ࠴࠼ߢ޽ࠆ‫ޕ‬ㆶరဳࠣ࡞࠲࠴ࠝࡦߩวᚑߦߪ㧞⒳㘃ߩ㉂⚛߇ᔅⷐߢ޽ࠆ‫߹ޕ‬ߕࠪࠬ࠹ࠗࡦߣࠣ࡞࠲ࡒࡦ㉄߇ǫ-ࠣ࡞࠲ࡒ࡞ࠪࠬ࠹ࠗPrimer㧘30 pM Reverse Primer㧘0.8 U Paq5000TM DNApolymerase)ࠍᷝടߒ㧘94͠/30 ⑽㧘60͠/35 ⑽㧘72͠/30⑽ࠍ 1 ࠨࠗࠢ࡞ߣߒߚ PCR ෻ᔕࠍ 45 ࠨࠗࠢ࡞ⴕߞߚ(2720 ࠨ࡯ࡑ࡞ࠨࠗࠢ࡜࡯/Applied Biosystems)‫ޕ‬෻ᔕ⚳

ੌᓟ㧘PCR Ⴧ᏷↥‛ࠍ 2%ࠕࠟࡠ࡯ࠬࠥ࡞ߦଏߒ㧘㔚᳇ᵒേࠍⴕߞߚ‫࠼ࠗࡑࡠࡉࡓ࠙ࠫ࠴ࠛޕ‬ᨴ⦡ߒߚ PCR Ⴧ᏷↥‛ࠍ UV ࠻࡜ࡦࠬࠗ࡞ࡒࡀ࡯࠲࡯ߦࠃߞߡ⏕⹺ߒߚ‫ޕ‬ࡦวᚑ㉂⚛(EC.6.3.2.2)ߦࠃߞߡ⚿วߒ,ߐࠄߦࠣ࡞࠲࠴

PCR ෻ᔕߦ↪޿ࠆ Primer ࠍᢙ⒳㘃⸳⸘ߒ㧘ቢో㐳 mRNAࠝࡦวᚑ㉂⚛(EC.6.3.2.3)߇ࠣ࡝ࠪࡦࠍ⚿วߐߖ,ࠣ࡞࠲ߩჇ᏷߇ᦨ߽⦟ᅢߥ⚵ߺวࠊߖࠍ⸃ᨆߦ↪޿ߚ‫ߚ߹ޕ‬㧘࠴ࠝࡦ߇วᚑߐࠇࠆ‫ޕ‬ǫ-ࠣ࡞࠲ࡒ࡞ࠪࠬ࠹ࠗࡦวᚑ㉂⚛

12 ⒳㘃ߩ exon ࠍⶄᢙหᤨߦჇ᏷ߔࠆࠃ߁ߦ primer ࠍ⸳߇㧞ߟߩ࠲ࡦࡄࠢ⾰ߩⶄว૕ߢ޽ࠆߩߦኻߒ㧘ࠣ࡞࠲࠴
ࠝࡦวᚑ㉂⚛ߪන⁛ߩ࠲ࡦࡄࠢ⾰ߢ޽ࠆߚ߼㧘ߘߩᄌ⇣ߪ⋥ធࠣ࡞࠲࠴ࠝࡦวᚑߦᓇ㗀ߔࠆߣ⠨߃ࠄࠇࠆ‫࡞ࠣޕ‬࠲ ࠴ ࠝ ࡦ ว ᚑ ㉂ ⚛ ࠍ ࠦ ࡯ ࠼ ߔ ࠆ ㆮ વ ሶ (glutathione
synthetase㧦GSS)ߪ㧘ࡅ࠻ߢߪ 20q11.2 ߦ૏⟎ߒ㧘12 ୘ߩexon ߆ࠄ 474 ୘ߩࠕࡒࡁ㉄ࠍ⠡⸶ߔࠆ1㧘2)‫ޕ‬ᚒ‫࡞ࠣߪޘ‬࠲࠴ࠝࡦวᚑ㉂⚛ㆮવሶߦ⌕⋡ߒ㧘ߘߩ mRNA ߩᄌ⇣⸃ᨆߩ⚿ᨐ㧘ቢో㐳 mRNA ߩઁߦ exon4 ߅ࠃ߮ 5 ࠍᰳᄬߔࠆ splicing variant ࠍ⊒⷗ߒߚߩߢߎߎߦႎ๔ߔࠆ‫ޕ‬
*1㧦Ꮏቇ⎇ⓥ⑼ᔕ↪ൻቇኾ᡹ඳ჻೨ᦼ⺖⒟*2㧦‛⾰↢๮ℂᎿቇ⑼ഥᢎ
*3

㧦‛⾰↢๮ℂᎿቇ⑼ಎᢎ᝼(hisatomi@st.seikei.ac.jp)ቯߒߚ‫ޕ‬Ⴧ᏷ߐࠇߚ PCR ↥‛ࠍ♖⵾ᓟ㧘BigDye Terminator
Cycle Sequencing Ready Reaction Kit (Applied Biosystems)ߦ ࠃ ࠅ ᮡ ⼂ ߒ 㧘 ABI PRISM 3130 Genetic Analyzer(Applied Biosystems)ߦࠃࠅႮၮ㈩೉ࠍ᳿ቯߒߚ‫ޕ‬᳿ቯߒߚ Ⴎ ၮ ㈩ ೉ ࠍ National Center for Biotechnology
Information ߩ࠺࡯࠲ࡌ࡯ࠬ(www.ncbi.nlm.nih.gov/)਄ߩ
GSS mRNA ߣᲧセߒ㧘ᄌ⇣ࠍหቯߒߚ‫ޕ‬㧟㧚⚿ ᨐ Huh-7 ↱᧪ cDNA ࠍ↪޿ߡ GSS mRNA ࠍჇ᏷ߒߚ⚿ᨐ㧘exon4 ߆ࠄ exon9 ߹ߢࠍ฽߻㗔ၞ(842 bp)ࠍჇ᏷ߒߚ

႐วߦ㧘⚂ 330 bp ⍴޿Ⴧ᏷↥‛߇⏕⹺ߐࠇߚ‫ߩߎޕ‬⍴޿㧙㧙㗔ၞߩᰳᄬࠍ⏕⹺ߒߚߦㆊ߉ߕ㧘exon4 ߅ࠃ߮ exon5 ࠍჇ᏷↥‛ࠍࠕࠟࡠ࡯ࠬࠥ࡞߆ࠄಾࠅ಴ߒ㧘Ⴎၮ㈩೉ࠍ᳿
ቯߒߚߣߎࠈ㧘exon4 ߅ࠃ߮ exon5 ߇ቢోߦᰳᄬߒߚ

ᰳᄬߔࠆᄌ⇣૕߇ mRNA ߣߒߡᯏ⢻ߔࠆߩ߆㧘޽ࠆ޿ߪvariant ߢ޽ࠆߎߣ߇⸽᣿ߐࠇߚ(Figure 1)‫ ߩઁޕ‬exon4 ߅࠲ࡦࡄࠢ⾰߇⠡⸶ߐࠇߡ޿ࠆ߆ߥߤߩ⺞ᩏߪᧂታᣉߢ޽

ࠃ߮ exon5 ࠍ฽߻ PCR ↥‛ߢ߽㧘ห᭽ߦ⍴޿Ⴧ᏷↥‛߇ࠆ‫ߚ߹ޕ‬㧘ᰳᄬ mRNA ߩሽ࿷Ყ߿⚵❱ߦ߅ߌࠆಽᏓߥߤ

⏕⹺ߐࠇߚ‫৻ޕ‬ᣇ㧘ߎࠇࠄ exon ࠍ฽߹ߥ޿㗔ၞࠍჇ᏷ߒ

߽ᧂ⸃᣿ߢ޽ࠆ‫੹ޕ‬ᓟ㧘ߎࠇࠄ⺖㗴ࠍ⸃᳿ߒ㧘ᧄ⎇ⓥߢ

ߚ PCR ↥‛ߢߪ㧘ᄌ⇣߇੍᷹น⢻ߥჇ᏷↥‛ߪ⏕⹺ߐࠇ⊒⷗ߒߚᄌ⇣ mRNA ߩᯏ⢻⸃ᨆࠍታᣉߔࠆ੍ቯߢ޽ࠆ‫ޕ‬ ᧄ⎇ⓥߢ⊒⷗ߒߚ GSS alternative splicing variant ߪ㧘ᧄߥ߆ߞߚ‫ޕ‬᧪ߥࠄ mRNA ߩᚑᾫㆊ⒟ߢ nonsense-mediated mRNA

decay system ߦࠃࠅಽ⸃ߐࠇࠆߪߕߢ޽ࠆ‫ޕ‬ᄌ⇣ mRNAߩሽ࿷ߪ㧘ߘߩ system ߆ࠄߩ࿁ㆱࠍᗧ๧ߒ㧘㕖Ᏹߦ⥝๧ᷓ޿‫ ߩߎޕ‬GSS alternative splicing variant ߪ㧘ቢో㐳mRNA ߆ࠄ⠡⸶ߐࠇࠆቢో㐳࠲ࡦࡄࠢ⾰ࠃࠅ߽ 111 ୘ߩ(KIWTG5GSWGPEGCPCN[UKUQHVJGVTCPUETKRVDGCTKPIVJGFGNGVKQPQHDQVJGZQPCPFGZQPQHVJG)55IGPG
6JG UGSWGPEG UJQYU VJG KPHTCOG FGNGVKQP
EQTTGURQPFKPIVQVJGEQORNGVGNQUUQHGZQPUCPF
5GSWGPEG CPCN[UGU YGTG RGTHQTOGF CU FGUETKDGF KP
/CVGTKCNUCPFOGVJQFUࠕࡒࡁ㉄߇ᰳᄬߒߡ޿ࠆ‫ࠅ߹ߟޕ‬㧘࠲ࡦࡄࠢ⾰ߩ N ᧃ┵߿ C ᧃ┵ߪ߽ߜࠈࠎ㧘ᰳᄬߒߚࠕࡒࡁ㉄㈩೉એᄖߪቢోߦ⛽ᜬߐࠇߡ޿ࠆ‫ߪࠇߎޕ‬㧘Dominat Negative Inhibitorߣߒߡᯏ⢻ߔࠆߚ߼ߦ㕖Ᏹߦᅢㇺวߢ޽ࠆ‫੹ޕ‬ᓟ㧘ߐ߹ߑ߹ߥ⸃ᨆߦࠃࠅ㧘ߘߩࠪࠣ࠽࡞વ㆐⚻〝߽หቯߔࠆ੍ቯߢ޽ࠆ‫ޕ‬㧠㧚⠨ ኤ     ෳ⠨ᢥ₂ ࠣ࡞࠲࠴ࠝࡦߪ⢄⤳߿⣢⤳㧘⿒ⴊ⃿ߥߤߦ㜞Ớᐲߦሽ࿷ߒ㧘⚦⢩ౝߢߩ࡟࠼࠶ࠢࠬ⁁ᘒߩ೙ᓮࠍᜂߞߡ޿ࠆ‫ޕ‬1) Shi, Z.-Z., Habib, G. M., Rhead, W. J., Gahl, W. A., He,X., Sazer, S., Lieberman, M. W. : Mutations in theߘߩߚ߼㧘ࠣ࡞࠲࠴ࠝࡦߩᰳਲߪ㧘හᐳߦ࡟࠼࠶ࠢࠬ⁁glutathione synthetase gene cause 5-oxoprolinuria. Natureᘒߩ፣უࠍ᜗߈㧘⚦⢩៊்ߩᒁ߈㊄ߦ߽ߥࠆ‫ߞ߇ߚߒޕ‬Genet. 14: 361-365, 1996.

ߡ㧘 ࠣ࡞࠲࠴ࠝࡦࠍวᚑߔࠆǫ-ࠣ࡞࠲ࡒ࡞ࠪࠬ࠹ࠗࡦ2) Webb, G. C., Vaska, V. L., Gali, R. R., Ford, J. H., Board,วᚑ㉂⚛߅ࠃ߮ࠣ࡞࠲࠴ࠝࡦวᚑ㉂⚛ߩᵴᕈൻߪᕡᏱ⊛P. G. : The gene encoding human glutathione synthetaseߢ޽ࠆᔅⷐ߇޽ࠆ‫ޕ‬GSSㆮવሶߩᄌ⇣ߦࠃࠆࠣ࡞࠲࠴ࠝ

(GSS) maps to the long arm of chromosome 20 at bandࡦวᚑ㉂⚛ߩࠕࡒࡁ㉄ᄌ⇣(ᵴᕈૐਅ)ߦࠃࠅǫ-ࠣ࡞࠲ࡒ

11.2. Genomics 30: 617-619, 1995.

࡞ࠪࠬ࠹ࠗࡦ߇ၮ⾰ߣߒߡ૶↪ߢ߈ߥߊߥࠆߣ㧘ㆊ೾ߥ3) Mohler, D. N., Majerus, P. W., Minnich, V., Hess, C. E.,

ǫ-ࠣ࡞࠲ࡒ࡞ࠪࠬ࠹ࠗࡦ߆ࠄวᚑߐߖࠆࡇࡠࠣ࡞࠲ࡒGarrick, M. D. : Glutathione synthetase deficiency as aࡦ㉄ߩዩ߳ߩឃᴭߦ⿠࿃ߔࠆ5́ࠝࠠ࠰ࡊࡠ࡝ࡦዩ∝(ࡇࡠࠣ࡞࠲ࡒࡦ㉄ዩ∝)߇⊒∝ߔࠆ3㧘4)

cause of hereditary hemolytic disease. New Eng. J. Med.

‫㉄ࡦࡒ࠲࡞ࠣࡠࡇޕ‬ዩ∝ߪᏱᨴ⦡૕ᕈഠᕈㆮવߩઍ⻢ᕈࠕࠪ࠼࡯ࠪࠬߢ޽ࠅ㧘4) Ristoff, E., Mayatepek, E., Larsson, A. : Long-termᖚ⠪ߩࠣ࡞࠲࠴ࠝࡦวᚑ㉂⚛ᵴᕈߩૐਅ߇ႎ๔ߐࠇߡ޿

clinical outcome in patients with glutathione synthetase3㧘4)ࠆ283: 1253-1257, 1970.

‫ߦ߁ࠃߩߎޕ‬㧘ࠣ࡞࠲࠴ࠝࡦวᚑ㉂⚛ࠍࠦ࡯࠼ߔࠆdeficiency. J. Pediat. 139: 79-84, 2001.

GSSㆮવሶߩᄌ⇣ߪ㧘ࠣ࡞࠲࠴ࠝࡦᷫዋߦࠃࠆ᛫㉄ൻ⢻ߩૐਅࠍ⺃⊒ߔࠆߛߌߢߥߊ㧘⋥ធ∔ᖚࠍ⊒∝ߐߖࠆߎߣ߇᣿ࠄ߆ߢ޽ࠆ‫⎇ᧄޕ‬ⓥߢ⊒⷗ߐࠇߚexon4߅ࠃ߮
exon5ߩᰳᄬGSSߪ㧘ᓥ᧪⊒⷗ߐࠇߡ޿ߚᄌ⇣ߣߪోߊ⇣ߥࠆߚ߼㧘ߘߩᯏ⢻⸃ᨆ߿࠲ࡦࡄࠢ⾰ಽᏓߩ⸃᣿ߦࠃࠅ㧘ᣂߚߥ∔ᖚߣߩ㑐ㅪ߇␜ໂߐࠇࠆน⢻ᕈ߇㜞޿‫ޕ‬ ᧄ⎇ⓥߢ⊒⷗ߒߚᄌ⇣૕ߩᰳᄬ㗔ၞߪࠣ࡞࠲ࡒࡦวᚑ
motif(202aa-302aa)ߩ⋥೨ߢ޽ࠅ motif ߇ᔅⷐߥ┙૕᭴ㅧࠍ⛽ᜬߔࠆߚ߼ߦ㊀ⷐߥ㗔ၞߢ޽ࠆߣ⠨߃ࠄࠇࠆ‫ᤨ⃻ޕ‬ὐߢߪ㧘PCR ෻ᔕߢჇ᏷ߐࠇࠆ exon3 ߆ࠄ exon8 ߹ߢߩ㧙㧙