[국내논문]묵납자루 (Acheilognathus signifer; Cyprinidae) metallothionein 유전자의 클로닝 및 특징 분석 Molecular Characterization of Metallothionein Gene of the Korean Bitterling Acheilognathus signifer (Cyprinidae)원문보기
한반도 고유종인 묵납자루(Acheilognathus signifer)로부터 metallothionein(MT) 유전자를 분리하고 그 유전자 구조와 발현 특징을 분석하였다. 묵납자루 MT cDNA는 20개의 시스테인(cysteines)을 포함한 60개의 아미노산을 암호화하고 있었고, 이들 시스테인 잔기들의 위치는 잉어목 어류에서 잘 보전되어 있었다. 묵납자루 MT 유전자는 3개의 exon과 2개의 intron으로 구성되어 있었으며 intron영역은 A/T조성 빈도가 높았다. 생물정보 분석법을 통해 묵납자루 MT 유전자의 프로모터 영역은 중금속 조절 빛 스트레스/면역관련 조절에 관련한 다양한 전사 조절인자들의 부착 위치들이 보유하고 있는 것으로 예측되었다. Real-timeRT-PCR 분석법을 이용한 묵납자루 MT mRNA의 조직 별 발현 수준을 조사한 결과, 난소와 장 조직에서의 발현 수준이 가장 높았으며 성장과 근욕 조직에서의 발현 수준이 가장 낮은 것으로 확인되었다. 구리를 이용한 중금속 노출 실험(구리 농도 $0.5\;{\mu}M$을 이용, 48 시간 동안 침지 처리)을 통하여 간 조직에서 MT mRNA 발현이 가장 많이 유도되었고(3.5배 이상), 비장, 신장 및 아가미에서도 유의적인 발현양의 증가(1.5~2.5배)가 관찰되었다. 그러나 뇌 및 장 조직에서는 MT 발현양의 변화가 없었다. 본 연구 결과는 향후 멸종위기 고유종인 묵납자루의 중금속 관련 스트레스 연구에 유용한 기초 자료를 제공할 수 있으리라 기대된다.
한반도 고유종인 묵납자루(Acheilognathus signifer)로부터 metallothionein(MT) 유전자를 분리하고 그 유전자 구조와 발현 특징을 분석하였다. 묵납자루 MT cDNA는 20개의 시스테인(cysteines)을 포함한 60개의 아미노산을 암호화하고 있었고, 이들 시스테인 잔기들의 위치는 잉어목 어류에서 잘 보전되어 있었다. 묵납자루 MT 유전자는 3개의 exon과 2개의 intron으로 구성되어 있었으며 intron영역은 A/T조성 빈도가 높았다. 생물정보 분석법을 통해 묵납자루 MT 유전자의 프로모터 영역은 중금속 조절 빛 스트레스/면역관련 조절에 관련한 다양한 전사 조절인자들의 부착 위치들이 보유하고 있는 것으로 예측되었다. Real-time RT-PCR 분석법을 이용한 묵납자루 MT mRNA의 조직 별 발현 수준을 조사한 결과, 난소와 장 조직에서의 발현 수준이 가장 높았으며 성장과 근욕 조직에서의 발현 수준이 가장 낮은 것으로 확인되었다. 구리를 이용한 중금속 노출 실험(구리 농도 $0.5\;{\mu}M$을 이용, 48 시간 동안 침지 처리)을 통하여 간 조직에서 MT mRNA 발현이 가장 많이 유도되었고(3.5배 이상), 비장, 신장 및 아가미에서도 유의적인 발현양의 증가(1.5~2.5배)가 관찰되었다. 그러나 뇌 및 장 조직에서는 MT 발현양의 변화가 없었다. 본 연구 결과는 향후 멸종위기 고유종인 묵납자루의 중금속 관련 스트레스 연구에 유용한 기초 자료를 제공할 수 있으리라 기대된다.
Genetic determinant for metallothionein (MT), a cysteine-rich protein playing essential roles in metal detoxification and homeostasis, was characterized in the Korean bitterling (Acheilognathus signifer, Cyprinidae), an endemic fish species. The full-length A. signifer MT (AsMT) cDNA (551 bp) is com...
Genetic determinant for metallothionein (MT), a cysteine-rich protein playing essential roles in metal detoxification and homeostasis, was characterized in the Korean bitterling (Acheilognathus signifer, Cyprinidae), an endemic fish species. The full-length A. signifer MT (AsMT) cDNA (551 bp) is composed of a single open-reading frame (ORF) to encode a polypeptide of 60 amino acids containing 20 cysteine residues whose positions are conserved in most cypriniform MTs. At the genomic level, the AsMT (2,593 bp spanning the 5'-flanking region to the 3'-untranslated region) represented a conserved tripartite (three exons interrupted by two introns) structure with AT-rich introns. The upstream regulatory region (-1,914 bp from the ATG initiation codon) of AsMT displayed various sites and motifs for transcription factors involved in the metal-mediated regulation and stress/immune responses. The AsMT transcript was ubiquitously detected in various organs with variable expression levels, where the ovary and intestine showed the highest expression, while the heart and skeletal muscle represented the lowest level. During an exposure to copper (immersion in $0.5\;{\mu}M$ Cu for 48 h), the levels of AsMT transcripts were significantly elevated in the liver (more than 3.5-fold), moderately in the gill, kidney, and spleen (ranging from 1.5- to 2.5-fold), and barely in the brain and intestine. Results of this study could form a useful basis to explore the metal-related stress physiology of this endangered fish species.
Genetic determinant for metallothionein (MT), a cysteine-rich protein playing essential roles in metal detoxification and homeostasis, was characterized in the Korean bitterling (Acheilognathus signifer, Cyprinidae), an endemic fish species. The full-length A. signifer MT (AsMT) cDNA (551 bp) is composed of a single open-reading frame (ORF) to encode a polypeptide of 60 amino acids containing 20 cysteine residues whose positions are conserved in most cypriniform MTs. At the genomic level, the AsMT (2,593 bp spanning the 5'-flanking region to the 3'-untranslated region) represented a conserved tripartite (three exons interrupted by two introns) structure with AT-rich introns. The upstream regulatory region (-1,914 bp from the ATG initiation codon) of AsMT displayed various sites and motifs for transcription factors involved in the metal-mediated regulation and stress/immune responses. The AsMT transcript was ubiquitously detected in various organs with variable expression levels, where the ovary and intestine showed the highest expression, while the heart and skeletal muscle represented the lowest level. During an exposure to copper (immersion in $0.5\;{\mu}M$ Cu for 48 h), the levels of AsMT transcripts were significantly elevated in the liver (more than 3.5-fold), moderately in the gill, kidney, and spleen (ranging from 1.5- to 2.5-fold), and barely in the brain and intestine. Results of this study could form a useful basis to explore the metal-related stress physiology of this endangered fish species.
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문제 정의
In line with our long-term goal to provide fine molecular biomarkers in order to aid the conservation activities for A. signifer, the objective of this study was to is이아e and characterize the genetic determinant of the metallothionein biomarker from this fish species.
제안 방법
Six-randomly-chosen recombinant clones per each PCR band were subjected to sequencing analysis at both directions. A eontinu셔us sequence of A. signifer MT cDNA containing the full-length open reading frame (ORF) was assembled in a contig using the sequence editing software, Sequencher (Gene Codes Cor., Ann Arbor, MI, USA), and the sequence was confirmed again by the RT-PCR isolation of fulLlength ORF from the whole body total R.NA using AsMTc FW and AsMTc RV primers.
Two successive PCR amplifications were performed with two reverse primers, AsMT-GW 1 and AsMT-GW2, which were respectively paired with API and AP2 forward adaptor primers provided in the kit (Clontech). Based on the contiguous sequence from the first genome walking, the second round of walking was conducted with two primer pairs, APl/AsMTGW3 and AP2/AsMT-GW4, in order to obtain the distal part of the 5'-flanking upstream region. A continuous fragment of the S'-upstream region was amplified again from the genomic DNA using the two PCR primers ASMTp-lF and ASMTp-lR, and its seq나ence was confirmed.
Four kinds of genome walking libraries were constructed with DraY-, EcoRV-, Pvull- or SwI-digested genomic DNA. Two successive PCR amplifications were performed with two reverse primers, AsMT-GW 1 and AsMT-GW2, which were respectively paired with API and AP2 forward adaptor primers provided in the kit (Clontech). Based on the contiguous sequence from the first genome walking, the second round of walking was conducted with two primer pairs, APl/AsMTGW3 and AP2/AsMT-GW4, in order to obtain the distal part of the 5'-flanking upstream region.
대상 데이터
html). Potential transcription factor binding sites or motifs were predicted using the Transcription Element Search System (http:// www.cbil.upenn.edu/cgi-bin/tess/tess?RQ=WELCOME)and TFSEARCH (http://www.cbrc.jp/research/db/TFSE ARCH.html).
데이터처리
Difference in the basal expression levels and the tissuedependent inducibility of MT transcripts among tissues were tested with ANOVA followed by Duncane multiple range tests using the SPSS software (ver. 10.1.3). Difference was considered to be significant when P<0.
이론/모형
gov) using the BLASTx or BLASTp options. Multiple sequence alignment of deduced amino acid seq나ence with representative orthologs from Cypriniformes was performed using the ClustalW (http ://align .genome .jp/). Predicted molecular weight (Mw) with the theoretical isoelectric point (pl) value was estimated using the ProtParam tool (http:// www.
jp/). Predicted molecular weight (Mw) with the theoretical isoelectric point (pl) value was estimated using the ProtParam tool (http:// www.expasy.org/tools/protparam.html). Potential transcription factor binding sites or motifs were predicted using the Transcription Element Search System (http:// www.
Based on the standard curves from MT and 18S rRNA genes, PCR efficiency for each gene was calculated. The relative expressions of MT transcripts across tissues were estimated based on the normalization against its own level of 18S rRNA expression using the comparative CT method (Schmittgen and Livak, 2008). Triplicate assays were carried out in an independent fashion.
성능/효과
, 2005, 2008; Scudiero et al, , 2005). From the m나Itiple sequence alignment with seventeen representative orthology from ten cypriniform species [A. signifer (abbreviation AC in Fig. 1), Cyprinus carpio (CC), Rutilus rutilus (RR), Gobio gobio (GG), Carassius cuvieri (CCu), Danio rerio (DR), Carassius auratus (CA), Barbatula barbatula (BB), Hemibarbus mylodon (HM) and Misgurnus mizolepis (MM)], nineteen of twenty Cys residues were clearly conserved in all other cypriniform species examined (Fig. 1). Only the exception was substitution of the 9th Cys residue to glycine (Gly), which was found in the zebrafish MT-IIB.
However the Cu exposure stimulated the expression of AsMT transcripts in several tissues. Of the six tissues tested, brain and intestine didn't show any alteration of the MT transcripts in response to the Cu exposure, whereas all other four tissues revealed the significant induction of MT transcripts. The induced amount of MT transcripts (i.
Only the exception was substitution of the 9th Cys residue to glycine (Gly), which was found in the zebrafish MT-IIB. The AsMT exhibited a considerable sequence identity (ranging from 93 to 100%) with its orthologs at both a- and p-domains, in which the highest sequence homology at protein level was found with the common carp (Cyprinus carpio) MT~2.
후속연구
During an acute exposure experiment using Cu, AsMT transcripts were significantly induced in the liver, moderately in the kidney and spleen, and hardly in brain and intestine. Results from this study would be a good start point to provide a useful basis to develop future experiments with MT gene and protein for addressing the metalcaused toxicity and stress in this fish species.
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