- cloned from the anterior horn tissues
1
Dorfin containing RING-finger and IBR motifs is a n E3 ubiquitin ligase that is localized in Lewy b odies, a characteristic neuronal inclusion in Par kinson’s disease brains. The Dorfin gene located on human chromosome 8q22.2 has showed 4.4 kb tran script and expressed ubiquitously in various tiss ues. Here we found its alternatively spliced tra nscript variants which derived from MaLR (mammali an LTR-retrotransposon) insertion. The MaLR-deri ved promoter transcripts are detected as two diff erent types in all tissues examined, while breast tissue only showed three variant types. Reporter gene assay of the promoter activity of MaLR eleme nt on Dorfin gene indicated good activity in huma n colon carcinoma cells (HCT-116). These finding s suggest that the MaLR element acquired the role of transcriptional regulation of Dorfin gene in v arious human tissues during primate evolution. . - cloned from the anterior horn tissues 분분 분분 genome transcriptome proteome genome 분분분분분 분분분 분분분분분 분분분 cDNA 분분분분 northern blot 분분 분분분분 분분 ALS 분분분 분분분 분분분 분분 E3 activity 분 분분 dorfin 분 분분분분 분분분분 분분분분분분분분분 분분분 분분 RBRfamily 분 분분분분 parkin 분분 homology 분분 분분분분분분분 분분분 분분 (VCP 분분 ) CaR 분 degradation 분 분분분 분분 분분 NM_01543 5.3 Parkinson’s disease Amyotrophic lateral sclerosis Other region 16% Gene-related Sequence 36% LINE 20% SINE 13% Coding sequence 3% Pseudogene 1% HERV element 8% DNA element 3% RT-PCR Realtime RT-PCR Cloning & Sequencing Promoter assay Sin HS, Huh JW, Kim DS, Kang DW, Min DS, Kim TH, Ha HS, Kim HH, Lee SY, Kim HS. 2006. Transcriptional control of the HERV-H LTR element of the GSDML gene in human tissues and canc Arch. Virol. [Epub ahead of print] Hishikawa N, Niwa J, Doyu M, Ito T, Ishigaki S, Hashizume Y, Sobue G. 2003. Dorfin localizes to the ubiquitylated inclusions in Parkinson's disease, dementia with Lewy bodi system atrophy, and amyotrophic lateral sclerosis. Am J Pathol. 163(2):609-619. Ma r ker Adr e nal g la n d A d u l t brain F e t a l brain F e ta l li v e r Heart K i dn e y Live r L ung B o n e marr o w C erebe l lu m S ke l e tal mu s cle Ma r ke r Pl ac e nta S p i na l c o rd T e s tis T h ymus T hyro i d T r a c hea Ute rus Prostat e Dorfin Gapdh 494 bp 195 bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Breast (N) Breast (C) Marker AAGGGAGCTAAATGGCGGGGTGGATGGAATTGCAAGTATTGAAAGTATAC R L E G N G D V I G A R L E G N G D V I G A I S GCACGGCCCAGCTGGATTTTGGACTTCTGGCCTCCAGAACTAGACAGGGC CTCACTGTGTCACCCAGGGTGGAATACAGTGGTGTGATCATAGCTCACTG CAGCCTGGAATTCCTGGGCTCAAGCAACCCTGCCACCTCAGCCTTCCAAG TAGCTAGGACTACAGAACATCCATGATAGCAGTCTTCTGTAAATCGAACT TTTCAAGAATTCTCTGAAGGAACCAAGTAGGATATTCTTACATCATGACT TAATGTGAATGCAAGAACAAGAAATAGGTTTTATCTCTAAATATAATGAA GGGCTGTGTGTAAACACTGACCCTGTCTCAATTCTAACAAGCATTTTAGA CATGAGTTTACATCGGCAAATGGGTTCAGATCGAGATCTTCAGTCCTCTG CTTCATCTGTGAGCTTGCCTTCAGTCAAAAAGGCACCCAAAAAAAGAAGA ATTTCAATAGGCTCCCTGTTTCGGAGGAAAAAAGATAACAAACGTAAATC AAGGGAGCTAAATGGCGGGGTGGATGGAATTGCAAGTATTGAAAGTATAC AGAAGCTGGAAGAGTCAAAGGACACATTCTCCCCTCAAGCCCCAGTGGGA GCACGGCCCAGCTGGATTTTGGACTTCTGGCCTCCAGAACTAGACAGGGC CTCACTGTGTCACCCAGGGTGGAATACAGTGGTGTGATCATAGCTCACTG CAGCCTGGAATTCCTGGGCTCAAGCAACCCTGCCACCTCAGCCTTCCAAG TAGCTAGGACTACAGAACATCCATGATAGCAGTCTTCTGTAAATCGAACT TTTCAAGAATTCTCTGAAGGAACCAAGTAGGATATTCTTACATCATGACT TAATGTGAATGCAAGAACAAGAAATAGGTTTTATCTCTAAATATAATGAA GGGCTGTGTGTAAACACTGACCCTGTCTCAATTCTAACAAGCATTTTAGA CATGAGTTTACATCGGCAAATGGGTTCAGATCGAGATCTTCAGTCCTCTG CTTCATCTGTGAGCTTGCCTTCAGTCAAAAAGGCACCCAAAAAAAGAAGA ATTTCAATAGGCTCCCTGTTTCGGAGGAAAAAAGATAACAAACGTAAATC Primer S2 Primer AS2 M Q E Q E I G F S K I Y G N E C L N V T P D S V I T L I S L D M L S R H Q G M D S R L D S Q S A S S S V L S P K V K P A K K R R R S I G I S F L K R K N D R K K S I S S E I M L S R H Q G M D S R L D S Q S A S S S V L S P K V K P A K K R R R S I G I S F L K R K N D R K K S S E I AluJ element AGAAGCTGGAAGAGTCAAAGGACACATTCTCCCCTCAAGCCCCAGTGGGA 556 bp 430 bp 315 bp (A) (B) (C) 3 11 1 2 MaLR AluJ AluJ MaLR 2 10 1 AluJ MaLR 2 11 1 3 126 bp 115 bp 1) 2) 3) 1 2 3 0 0.01 0.02 0.03 0.04 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 MaLR-derived promoter transcript (556 bp) MaLR-derived pro moter transcript (430 bp) Relative Expres sion Bone marrow Cerebellum Kidney Lung Testis Reverse Forward Reverse Forward Control Relative Luciferase Activity (Fold of pGL-2 control) 0 2 4 6 8 10 12 14 16 18 20 22 Cos7 HCT116 CCCCACAAAA GATATGTTCA TGTCCTAATC CCCAGAATCT GCAAATGTTA TTTGGAAAAA GGGGT TTTGC AGATGTAATT AAGTTAAGAA TCTTGAGATA AGATCATCCT GGATTATCCA GGTAGCCTCA AAATCAAGTG ACAAGTGTCT TTGTAAGGGA CAAGTAGACC CATTACAGAG AAGACGACGC GCAG AAAAGG AGGAAGCAGT GTGCTCATGG AGGCGGAGAT TGGAGTGATG TAACCGCAAG CCGAGGAAT G CTTATAGTCA CCAGAAGCTG GAAGAGTCAA AGGACACATT CTCCCCTCAA GCCCCAGTGG GA GCACGGCC CAGCTGGATT TTGGACTTCT GGCCTCCAGA ACTGTAAGAG AAATGTCCAT TGTCTTA AGC CAACCAGTTT GTGGTAGTTT GTTACAGCAG CCCCAGGAAA CTACTA . GATA-1 GATA-2 Nkx2-5 Nkx2-5 Whn ELF-1 +1 TRANSCRIPTION START SITE In silico analysis of transcription binding sites of MaLR element H um an D og Pig C attle M ouse R at Chicken Zebrafish 100 100 100 42 77 0.05 100 77 42 100 100 100 Human Dog Pig Cattle Mouse Rat Zebrafish Chicken
description
Primer S2. (A). (B). AGAAGCTGGAAGAGTCAAAG GACACATTCTCCCCTCAAGC CCCAGTGGGA. AGAAGCTGGAAGAGTCAAAG GACACATTCTCCCCTCAAGC CCCAGTGGGA. Breast (C). - PowerPoint PPT Presentation
Transcript of - cloned from the anterior horn tissues
Dorfin containing RING-finger and IBR motifs is an E3 ubiquitin ligase that is localized in Lewy bodies, a characteristic neuronal inclusion in Parkinson’s disease brains. The Dorfin gene located on human chromosome 8q22.2 has showed 4.4 kb transcript and expressed ubiquitously in various tissues. Here we found its alternatively spliced transcript variants which derived from MaLR (mammalian LTR-retrotransposon) insertion. The MaLR-derived promoter transcripts are detected as two different types in all tissues examined, while breast tissue only showed three variant types. Reporter gene assay of the promoter activity of MaLR element on Dorfin gene indicated good activity in human colon carcinoma cells (HCT-116). These findings suggest that the MaLR element acquired the role of transcriptional regulation of Dorfin gene in various human tissues during primate evolution. .
- cloned from the anterior horn tissues
분류 연구
genome
transcriptome
proteome
genome 수준에서의 연구는이루어지지 않았음
cDNA 서열결정northern blot 으로 발현패턴 확인ALS 에서의 전사체 발현양 연구
E3 activity 의 확인dorfin 이 존재하는 위치확인신경퇴행성질병과의 관련성 연구RBRfamily 의 계통연구parkin 과의 homology 연구활성메카니즘에 관련된 연구 (VCP 관련 )CaR 의 degradation 에 미치는 영향 연구
NM_015435.3
Parkinson’s disease Amyotrophic lateral sclerosis
Other region16%
Gene-related Sequence
36%
LINE20%
SINE13%
Coding sequence3%
Pseudogene1%
HERV element8%DNA element
3%
RT-PCR
RealtimeRT-PCR Cloning &
Sequencing
Promoterassay
Sin HS, Huh JW, Kim DS, Kang DW, Min DS, Kim TH, Ha HS, Kim HH, Lee SY, Kim HS. 2006.Transcriptional control of the HERV-H LTR element of the GSDML gene in human tissues and cancer cells.Arch. Virol. [Epub ahead of print]
Hishikawa N, Niwa J, Doyu M, Ito T, Ishigaki S, Hashizume Y, Sobue G. 2003.Dorfin localizes to the ubiquitylated inclusions in Parkinson's disease, dementia with Lewy bodies, multiple system atrophy, and amyotrophic lateral sclerosis.Am J Pathol. 163(2):609-619.
Mar
ker
Adr
enal
gla
nd
Adu
lt br
ain
Fet
al b
rain
Fet
al li
ver
Hea
rtK
idne
yL
iver
Lun
g
Bon
e m
arro
wC
ereb
ellu
m
Skel
etal
mus
cle
Mar
ker
Pla
cent
a
Spin
al c
ord
Tes
tisT
hym
usT
hyro
id
Tra
chea
Ute
rus
Pro
stat
e
Dorfin
Gapdh
494 bp
195 bp
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
Bre
ast
(N)
Bre
ast
(C)
Mar
ker
AAGGGAGCTAAATGGCGGGGTGGATGGAATTGCAAGTATTGAAAGTATAC
R LE GN G DV IG A
R LE GN G DV IG A IS
GCACGGCCCAGCTGGATTTTGGACTTCTGGCCTCCAGAACTAGACAGGGC
CTCACTGTGTCACCCAGGGTGGAATACAGTGGTGTGATCATAGCTCACTG
CAGCCTGGAATTCCTGGGCTCAAGCAACCCTGCCACCTCAGCCTTCCAAG
TAGCTAGGACTACAGAACATCCATGATAGCAGTCTTCTGTAAATCGAACT
TTTCAAGAATTCTCTGAAGGAACCAAGTAGGATATTCTTACATCATGACT
TAATGTGAATGCAAGAACAAGAAATAGGTTTTATCTCTAAATATAATGAA
GGGCTGTGTGTAAACACTGACCCTGTCTCAATTCTAACAAGCATTTTAGA
CATGAGTTTACATCGGCAAATGGGTTCAGATCGAGATCTTCAGTCCTCTG
CTTCATCTGTGAGCTTGCCTTCAGTCAAAAAGGCACCCAAAAAAAGAAGA
ATTTCAATAGGCTCCCTGTTTCGGAGGAAAAAAGATAACAAACGTAAATC
AAGGGAGCTAAATGGCGGGGTGGATGGAATTGCAAGTATTGAAAGTATAC
AGAAGCTGGAAGAGTCAAAGGACACATTCTCCCCTCAAGCCCCAGTGGGA
GCACGGCCCAGCTGGATTTTGGACTTCTGGCCTCCAGAACTAGACAGGGC
CTCACTGTGTCACCCAGGGTGGAATACAGTGGTGTGATCATAGCTCACTG
CAGCCTGGAATTCCTGGGCTCAAGCAACCCTGCCACCTCAGCCTTCCAAG
TAGCTAGGACTACAGAACATCCATGATAGCAGTCTTCTGTAAATCGAACT
TTTCAAGAATTCTCTGAAGGAACCAAGTAGGATATTCTTACATCATGACT
TAATGTGAATGCAAGAACAAGAAATAGGTTTTATCTCTAAATATAATGAA
GGGCTGTGTGTAAACACTGACCCTGTCTCAATTCTAACAAGCATTTTAGA
CATGAGTTTACATCGGCAAATGGGTTCAGATCGAGATCTTCAGTCCTCTG
CTTCATCTGTGAGCTTGCCTTCAGTCAAAAAGGCACCCAAAAAAAGAAGA
ATTTCAATAGGCTCCCTGTTTCGGAGGAAAAAAGATAACAAACGTAAATC
Primer S2
Primer AS2
M Q E Q E I G F S KI Y
G
N E
CL NV T PD SV I TL IS L D
M LS RH Q GM DS R LD SQ S
A SS SV L SP KV K PA KK R R
R SI GI S FL KR K ND RK K S
IS SE I
M LS RH Q GM DS R LD SQ S
A SS SV L SP KV K PA KK R R
R SI GI S FL KR K ND RK K S
SE I
AluJ element
AGAAGCTGGAAGAGTCAAAGGACACATTCTCCCCTCAAGCCCCAGTGGGA
556 bp430 bp315 bp
(A) (B)
(C)
3 111 2
MaLR AluJ
AluJMaLR
2 101
AluJMaLR
2 111 3
126 bp 115 bp
1)
2)
3)
123
0
0.01
0.02
0.03
0.04
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
MaLR-derived promoter transcript (556 bp)
MaLR-derived promoter transcript (430 bp)
Rel
ativ
e E
xpre
ssio
n
Bone marrow
Cerebellum Kidney
Lung
Testis
Reverse
Forward
Reverse
Forward
Control
Relative Luciferase Activity
(Fold of pGL-2 control)
0 2 4 6 8 10 12 14 16 18 20 22
Cos7
HCT116
CCCCACAAAA GATATGTTCA TGTCCTAATC CCCAGAATCT GCAAATGTTA TTTGGAAAAA GGGG
TTTTGC AGATGTAATT AAGTTAAGAA TCTTGAGATA AGATCATCCT GGATTATCCA GGTAGCCT
CA AAATCAAGTG ACAAGTGTCT TTGTAAGGGA CAAGTAGACC CATTACAGAG AAGACGACGC
GCAGAAAAGG AGGAAGCAGT GTGCTCATGG AGGCGGAGAT TGGAGTGATG TAACCGCAAG CC
GAGGAATG CTTATAGTCA CCAGAAGCTG GAAGAGTCAA AGGACACATT CTCCCCTCAA GCCCC
AGTGG GAGCACGGCC CAGCTGGATT TTGGACTTCT GGCCTCCAGA ACTGTAAGAG AAATGTCCA
T TGTCTTAAGC CAACCAGTTT GTGGTAGTTT GTTACAGCAG CCCCAGGAAA CTACTA
.
GATA-1
GATA-2
Nkx2-5 Nkx2-5
Whn ELF-1
+1 TRANSCRIPTION START SITE
In silico analysis of transcription binding sites of MaLR element
Human
Dog
Pig
Cattle
Mouse
Rat
Chicken
Zebrafish
100
100
100
42
77
0.05
100
77
42
100
100
100
Human
Dog
Pig
Cattle
Mouse
Rat
ZebrafishChicken
