[국내논문]미호종개 metallothionein 유전자의 구조 및 중금속 노출과 고온 자극에 대한 MT mRNA의 발현 특징 분석 Gene Structure and Altered mRNA Expression of Metallothionein in Response to Metal Exposure and Thermal Stress in Miho Spine Loach Cobitis choii (Cobitidae; Cypriniformes)원문보기
멸종위기 어류 미호종개(Cobitis choii)로부터 중금속해독 단백질(metallothionein) 유전자를 분리, 클로닝하고 중금속 및 고온 스트레스에 대한 전사 발현 특정을 분석하였다. 미호종개 metallothionein는 gDNA, mRNA 및 아미노산 서열 모두에서 경골 어류 MT들의 구조적 특징을 잘 보전하고 있었으며, 생물정보분석을 통해 미호종개 MT 유전자 5'-upstream 영역은 중금속 조절, 면역 반응 및 온도 반응에 관여하는 다양한 전사 조절인자들의 부착 위치들을 포함하는 것으로 관찰되었다. 카드뮴(Cd), 구리(Cu), 니켈(Ni), 망간(Mn) 및 아연(Zn)을 이용한 침지 노출 실험(0.5 및 $1.0\;{\mu}M$; 24시간)에서 미호종개 MT mRNA 발현은 구리 및 카드뮴 처리군에서 가장 많이 유도되었고($1.0\;{\mu}M$ Cu 처리군에서 최대 10배), 망간 처리군에서는 비교적 적은 양의 MT 발현이 유도된 반면(2배), 아연 및 니켈 노출 군에서는 유의적인 MT 발현의 증감이 관찰되지 않았다. 또한 미호종개 MT 전사 발현은 고온 자극 ($25^{\circ}C$로부터 $31^{\circ}C$까지 증가)에도 민감하게 반응하는 것으로 나타나, $31^{\circ}C$ 도달시점에서 $25^{\circ}C$ 초기 MT mRNA 발현 수준보다 9배 높은 mRNA 발현이 관찰되었다. 본 연구 결과는 MT 기반의 유전자 발현 분석을 이용함으로써, 향후 멸종위기 어류 미호종개의 스트레스 반응 연구에 유용한 기초 자료를 제공할 수 있다고 기대된다.
멸종위기 어류 미호종개(Cobitis choii)로부터 중금속해독 단백질(metallothionein) 유전자를 분리, 클로닝하고 중금속 및 고온 스트레스에 대한 전사 발현 특정을 분석하였다. 미호종개 metallothionein는 gDNA, mRNA 및 아미노산 서열 모두에서 경골 어류 MT들의 구조적 특징을 잘 보전하고 있었으며, 생물정보분석을 통해 미호종개 MT 유전자 5'-upstream 영역은 중금속 조절, 면역 반응 및 온도 반응에 관여하는 다양한 전사 조절인자들의 부착 위치들을 포함하는 것으로 관찰되었다. 카드뮴(Cd), 구리(Cu), 니켈(Ni), 망간(Mn) 및 아연(Zn)을 이용한 침지 노출 실험(0.5 및 $1.0\;{\mu}M$; 24시간)에서 미호종개 MT mRNA 발현은 구리 및 카드뮴 처리군에서 가장 많이 유도되었고($1.0\;{\mu}M$ Cu 처리군에서 최대 10배), 망간 처리군에서는 비교적 적은 양의 MT 발현이 유도된 반면(2배), 아연 및 니켈 노출 군에서는 유의적인 MT 발현의 증감이 관찰되지 않았다. 또한 미호종개 MT 전사 발현은 고온 자극 ($25^{\circ}C$로부터 $31^{\circ}C$까지 증가)에도 민감하게 반응하는 것으로 나타나, $31^{\circ}C$ 도달시점에서 $25^{\circ}C$ 초기 MT mRNA 발현 수준보다 9배 높은 mRNA 발현이 관찰되었다. 본 연구 결과는 MT 기반의 유전자 발현 분석을 이용함으로써, 향후 멸종위기 어류 미호종개의 스트레스 반응 연구에 유용한 기초 자료를 제공할 수 있다고 기대된다.
Gene and promoter structures of metallothionein(MT) from Miho spine loach (Cobitis choii; Cypriniformes) were characterized, and the transcriptional responses to experimental exposures to heavy metals and heat stress were examined. The C. choii metallothionein displayed well-conserved features of te...
Gene and promoter structures of metallothionein(MT) from Miho spine loach (Cobitis choii; Cypriniformes) were characterized, and the transcriptional responses to experimental exposures to heavy metals and heat stress were examined. The C. choii metallothionein displayed well-conserved features of teleostean metallothioneins at gDNA, mRNA and amino acid levels. Bioinformatic analysis predicted that the C. choii MT regulatory region potentially possessed various motifs or elements targeted by various transcription factors associated with metal-coordinating regulation (e.g., metal transcription factor-1), immune responses (e.g., nuclear factor kappa B), and thermal modulations (e.g., heat shock factor). Acute heavy-metal exposures to 0.5 or $1.0\;{\mu}M$ of cadmium (Cd), copper (Cu), manganese (Mn), nickel (Ni) or zinc (Zn) showed that MT transcription was significantly stimulated by Cd (9.6-fold relative to non-exposed control) and Cu (10.4-fold), only moderately by Mn (2.4-fold), but hardly by Ni and Zn. Elevation of water temperature from $25^{\circ}C$ to $31^{\circ}C$ caused a rapid modulation of MT mRNAs toward upregulation to 9.5-fold; however, afterward the elevated mRNA level slightly decreased during further incubation at $31^{\circ}C$ for 6 h. Results from this study suggest that MT-based expression assay could be a useful basis for better understanding the metal- and/or heat-caused stresses in this endangered fish species.
Gene and promoter structures of metallothionein(MT) from Miho spine loach (Cobitis choii; Cypriniformes) were characterized, and the transcriptional responses to experimental exposures to heavy metals and heat stress were examined. The C. choii metallothionein displayed well-conserved features of teleostean metallothioneins at gDNA, mRNA and amino acid levels. Bioinformatic analysis predicted that the C. choii MT regulatory region potentially possessed various motifs or elements targeted by various transcription factors associated with metal-coordinating regulation (e.g., metal transcription factor-1), immune responses (e.g., nuclear factor kappa B), and thermal modulations (e.g., heat shock factor). Acute heavy-metal exposures to 0.5 or $1.0\;{\mu}M$ of cadmium (Cd), copper (Cu), manganese (Mn), nickel (Ni) or zinc (Zn) showed that MT transcription was significantly stimulated by Cd (9.6-fold relative to non-exposed control) and Cu (10.4-fold), only moderately by Mn (2.4-fold), but hardly by Ni and Zn. Elevation of water temperature from $25^{\circ}C$ to $31^{\circ}C$ caused a rapid modulation of MT mRNAs toward upregulation to 9.5-fold; however, afterward the elevated mRNA level slightly decreased during further incubation at $31^{\circ}C$ for 6 h. Results from this study suggest that MT-based expression assay could be a useful basis for better understanding the metal- and/or heat-caused stresses in this endangered fish species.
* AI 자동 식별 결과로 적합하지 않은 문장이 있을 수 있으니, 이용에 유의하시기 바랍니다.
문제 정의
The objective of this study is to isolate and characterize the genetic determinant of C. choii metallothionein at both mRNA and genomic levels in order to provide a basis for further development of A/T-based biomarker. For this, we characterized the full-length cDNA and genomic gene sequences of the C.
We thank Dr. In-Chul Bang, Soonchunhyang University for his kind providing the experimental fish specimens as well as for his critical comments on this study. This study was supported by a fund from the KoreaUkraine international cooperative research project (#2010- 00091) from National Research Foundation.
제안 방법
choii metallothionein at both mRNA and genomic levels in order to provide a basis for further development of A/T-based biomarker. For this, we characterized the full-length cDNA and genomic gene sequences of the C. choii MT, highlighted the structural characteristics of the 5z-flanking upstream regulatory region and examined the tran实ription시 alterations of MT in response to acute metal expos나res and heat stress.
In order to examine the modulation of MT gene in response to heat stress, a thermal treatment was performed. Forty fry (same-sized as above) were allocated into one of two identical tanks (50 L) containing 40 L of tap water at 25℃, and incubated further for 24 h in order to make the fish to ac시imate to the starting temperature.
From the contig sequence, a genomic DNA fragment from the 5'-upstream region to 3, Utr was pcR-aniplified again by using the primers ICMTp FW and ICMTp-RV, and then the sequence was confirmed. Putative transcription factor binding motifs was predicted using the Transcription Element Search System (TESS; http://www.cbiLupenn.edu/cgi-bin/tess/ tess?RQ = WELCOME) and Transcriptional Factor Search (TFSEARCH; http://www.cbrc.jp/research/db/TFSEA RCH.html).
대상 데이터
choii fry to various heavy metals were performed. Eight C. choii fry (average body wei응ht= 1.1 ±0.2 g) were allocated into one of two replicate tank동 (10 L) filled with 8 1.of well-aerated tap water (5.0 pm-filtered) containing 0.5 or 1.0 gM of cadmium (Cd), copper (Cu), manganese (Mn), nickel (Ni) or zinc (Zn). A non-exposed control group was also prepared identically except the administration of the heavy metals.
The C. choii MT cDNA was composed of 56 bp of 5'-UTR, 180 bp of si員읺e open reading frame (ORF) encoding 60 amino acids and 243 bp of 3'~UTR excluding the poly (A) + tail (Fig. 1; GenBank accession no. JF419523). Two putative poly쇼denylation signals (AATATA and AATAAA) were found at 153 bp and 16 bp prior to the poly(A) + tail, respectively, suggesting the potential processing of MT transcript variants with different 3'-UTR leri용ths (Cho et 사, .
데이터처리
Triplicate assays were performed in an independent fashion. Based on the scanning densitometry, differences in the relative expression levels among groups were assessed by analysis of variance (ANOVA) followed by Duncane multiple range tests. On the other hand, significant difference from the control levels was tested by Studente rtest.
Transcriptional modulation (fold-induction) of Cobitis choii MT during thermal elevation from 25℃ to 31℃ (TC/h), as assessed by semi-qeantitative RT-PCR analysis. Mean±SDs with different letters (a-d) are significantly different based on ANOVA followed by Duncan's multiple range tests at p V 0.05. Significant elevations of MT transcript levels from the basal level observed in the control group kept at the constant temperature of 25℃ are indicated by asterisks based on Studenf s r-test (P<0.
0 for 24 h) to Cd, Cu, Mn, Ni or Zn, as assessed by semi-quantitative RT-PCR analysis. Mean±SDs with different letters (a-e) are significantly different based on ANOVA followed by Duncan's multiple range tests at P<0.05. Significant elevation from the basal level observed in the non-exposed control group is indicated by asterisks based on Student's Mest (P<0.
이론/모형
Genomic organization (exon/intron organization) was compared to other teleost AfTgenes. Nucleotide composition analysis of each intron was done using the GENE BOY tool (http: //www.dnai .org/geneboy/index .html). Homology search against GenBaiik was performed using BLASTx or BLASTp option.
성능/효과
3). Of the five metals tested, most potent metals were Cd (8.2- and 9.6-fold induction of MTmRNA, respectively for 0.5 and 1.0 eM-treated groups when compared to basal level of non-exposed control) and Cu(5.1- and 10.4-fold), while fish exposed to Mn showed only the moderate activation of MT at both 0.5 and 1.0 μM concentrations (2.4- and 2.2-fold, respectively) (P<0.05). On the other hand, significant alteration was detected in neither Ni nor Zn irrespective of the metal concentrations (P> 0.
후속연구
, 2009). In addition, the possibility that tissuespecific modulation of the MT transcripts might be masked under the present assay condition because we performed the analysis with the total RNA samples from whole fry rather than independent adult tissues, which should be addressed in future experiment. Nevertheless, result from this study could support, at least in part, the recent claim on the need of revision of the classic dogma for the MT induction, in the sense of that Zn is not always an inducer for activating the MT transcription (Bi et al, 2006; Bourdineaud et al.
Result of this study proposes that MT could be a potential indicator to address the various cellular stresses in this endangered fish species, however also suggests that careful and extensive analyses should be followed in order to employ the MT as an environment-realistic biomarker to aid the conservation of this species. In particular, the assessment of MT modulation with more realistic doses of the heavy metals, which are expected potentially to be present in candidate recipient sites for the restoration of this species (i.e. the sites for re-introduction of the fishes), would be a valuable subject in future study. However for this assessment also, it is recommended that seasonal variation of MT gene expression caused by the change of water temperature should be taken together into account.
Although we haven't yet examined the expression pattern of antioxidant enzyme genes together with MT gene, the temperat탾reedependcnt upregulation of MT may be in accordance with its potential involvement in the antioxidant pathway to relieve the prooxidant stress arisen from the thermal elevation. On the other hand, the decline of MT transcript levels after reaching peak at 31℃ could be explained, in general, by the stabilized or acclimated process after the initial shock-phase, indicating future experiments are needed to examine if the MT mRNA levels could return to the basal level or not. It is widely agreed that metallothionein is a useful early warning indicator of the risks associated with heavy metal-mediat-ed toxicity (Rodrfguez-Ortega et 시/.
choii MT in response to experimental exposures with different heavy metals and heat stress were examined. Result of this study proposes that MT could be a potential indicator to address the various cellular stresses in this endangered fish species, however also suggests that careful and extensive analyses should be followed in order to employ the MT as an environment-realistic biomarker to aid the conservation of this species. In particular, the assessment of MT modulation with more realistic doses of the heavy metals, which are expected potentially to be present in candidate recipient sites for the restoration of this species (i.
참고문헌 (41)
Amiard, J.C., C. Amiard-Triquet, S. Barka, J. Pellerin and P.S. Rainbow. 2006. Metallothioneins in aquatic invertebrates: their role in metal detoxification and their use as biomarkers. Aquat. Toxicol., 76: 160-202.
Atif, F., M. Kaur, S. Yousuf and S. Raisuddin. 2006. In vitro free radical scavenging activity of hepatic metallothionein induced in an Indian freshwater fish, Channa punctatta Bloch. Chem. Biol. Interact., 162: 172-180.
Bang, I.C., W.J. Kim and I.R. Lee. 2009. Characterization of polymorphic microsatellite loci in the endangered Miho spine loach (Iksookimia choii) and cross-species amplification within the Cobitidae family. Mol. Ecol.Resour., 9: 281-284.
Bi, Y., G.X. Lin, L. Millecchia and Q. Ma. 2006. Superinduction of metallothionein I by inhibition of protein synthesis: role of a labile repressor in MTF-I mediated gene transcriptioin. J. Biochem. Mol. Toxicol., 20: 57-68.
Bourdineaud, J.P., M. Baudrimont, P. Gonzalez and J.L. Moreau. 2006. Challenging the model for induction of metallothionein gene expression. Biochimie, 88: 1787-1792.
Buckley, B.A., A.Y. Gracey and G.N. Somero. 2006. The cellular response to heat stress in the goby Gillichthys mirabilis: a cDNA microarray and protein-level analysis. J. Exp. Bio., 209: 2660-2677.
Chen, W.Y., J.A.C. John, C.-H. Lin, H.-F. Lin, S.-C. Wu, C.-H. Lin and C.-Y. Chang. 2004. Expression of metallothionein gene during embryonic and early larval development in zebrafish. Aquat. Toxicol., 69: 215-227.
Cho, Y.S., B.N. Choi, E.M. Ha, K.H. Kim, S.K. Kim, D.S. Kim and Y.K. Nam. 2005. Shark (Scyliorhinus torazame) metallothionein: cDNA cloning, genomic sequence, and expression analysis. Mar. Biotechnol., 7: 350-362.
Cho, Y.S., I.C. Bang, I.R. Lee and Y.K. Nam. 2009a. Hepatic expression of Cu/Zn-superoxide dismutase transcripts in response to acute metal exposure and heat stress in Hemibarbus mylodon (Teleostei: Cypriniformes). Fish. Aqua. Sci., 12: 179-184.
Cho, Y.S., S.Y. Lee, C.H. Noh, Y.K. Nam and D.S. Kim. 2006. Survey of genes responsive to long-term heat stress using a cDNA microarray analysis in mud loach (Misgurnus mizolepis) liver. Kor. J. Ichthyol., 18: 65-77.
Cho, Y.S., S.Y. Lee, K.H. Kim, S.K. Kim, D.S. Kim and Y.K. Nam. 2009b. Gene structure and differential modulation of multiple rockbream (Oplegnathus fasciatus) hepcidin isoforms resulting from different biological stimulations. Dev. Comp. Immunol., 33: 46-58.
Cho, Y.S., S.Y. Lee, K.-Y. Kim, I.C. Bang, D.S. Kim and Y.K. Nam. 2008. Gene structure and expression of metallothionein during metal exposures in Hemibarbus mylodon, Ecotoxicol. Environ. Saf., 71: 125-137.
Cho, Y.S., S.Y. Lee, K.-Y. Kim and Y.K. Nam. 2009c. Two metallothionein genes from mud loach Misgurnus mizolepis (Teleostei; Cypriniformes): Gene structure, genomic organization, and mRNA expression analysis. Comp. Biochem. Physiol. B Biochem. Mol. Biol., 153: 317-326.
Dragun, Z., M. Podrug and B. Raspor. 2009. Combined use of bioindicators and passive samplers for the assessment of the river water contamination with metals. Arch. Environ. Contam. Toxicol., 57: 211-220.
Falfushynska, H.I., L.L. Gnatyshyna, C.V. Priydun, O.B. Stoliar and Y.K. Nam. 2010. Variability of responses in the crucian carp Carassius carassius from two Ukrainian ponds determined by multi-marker approach. Ecotoxicol. Environ. Saf., 73: 1896-1906.
Ferencz, A. and E. Hermesz. 2009. Identification of a splice variant of the metal-responsive transcription factor MTF-1 in common carp. Comp. Biochem. Physiol. C Toxicol. Pharmacol., 150: 113-117.
Fu, Z., J. Guo, L. Jing, R. Li, T. Zhang and S. Peng. 2010. Enhanced toxicity and ROS generation by doxorubicin in primary cultures of cardiomyocytes from neonatal metallothionein-I/II null mice. Toxicol. In Vitro, 24: 1584-1591.
Gao, D., G.T. Wang, X.T. Chen and P. Nie. 2009. Metallothionein- 2 gene from the mandarin fish Siniperca chuatsi: cDNA cloning, tissue expression, and immunohistochemical localization. Comp. Biochem. Physiol. C Toxicol. Pharmacol., 149: 18-25.
Guo, R., H. Ma, F. Gao, L. Zhong and J. Ren. 2009. Metallothionein alleviates oxidative stress-induced endoplasmic reticulum stress and myocardial dysfunction. J. Mol. Cell. Cardiol., 47: 228-237.
He, P., M. Xu and H. Ren. 2007. Cloning and functional characterization of 5′-upstream region of metallothionein- I gene from crucian carp (Carassius cuvieri). Int. J. Biochem. Cell Biol., 39: 832-841.
Hermesz, E., M. Abraham and J. Nemcsok. 2001. Tissuespecific expression of two metallothionein genes in common carp during cadmium exposure and temperature shock. Comp. Biochem. Physiol. C Toxicol. Pharmacol., 128: 457-465.
Kalman, J., I. Riba, T. Angel DelValls and J. Blasco. 2010. Comparative toxicity of cadmium in the commercial fish species Sparus aurata and Solea senegalensis. Ecotoxicol. Environ. Saf., 73: 306-311.
Kim, K.-Y., S.Y. Lee, Y.S. Cho, I.C. Bang, D.S. Kim and Y.K. Nam. 2008. Characterization and phylogeny of two beta-cytoskeletal actins from Hemibarbus mylodon (Cyprinidae, Cypriniformes), a threatened fish species in Korea. DNA Seq., 19: 87-97.
Knapen, D., E.S. Redeker, I. Inacio, W. De Coen, E. Verheyen and R. Blust. 2005. New metallothionein mRNAs in Gobio gobio reveal at least three gene duplication events in cyprinid metallothionein evolution. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol., 140: 347-355.
Laity, J.H. and G.K. Andrews. 2007. Understanding the mechanisms of zinc-sensing by metal-response element binding transcription factor-1 (MTF-1). Arch. Biochem. Biophys., 463: 201-210.
Lee, S.Y., O. Stoliar and Y.K. Nam. 2010. Transcriptional alteration of two metallothionein isoforms in mud loach (Misgurnus mizolepis) fry during acute heavy metal exposure. Fish. Aqua. Sci., 13: 112-117.
Lee, Y.A., Y.E. Yun, Y.K. Nam and I.C. Bang. 2008. Genetic diversity of an endangered fish Iksookimia choii (Cypriniformes), from Korea as assessed by amplified fragment length polymorphism. Kor. J. Limnol., 41: 98-103.
Lin, C.H., J.A.C. John, L.W. Ou, J.C. Chen, C.H. Lin and C.Y. Chang. 2004. Cloning and characterization of metallothionein gene in ayu Plecoglossus altivelis. Aquat. Toxicol., 66: 111-124.
Ren, H., M. Xu, P. He, N. Muto, N. Itoh, K. Tanaka, J. Xing and M. Chu. 2006. Cloning of crucian carp (Carassius cuvieri) metallothionein-II gene and characterization of its gene promoter region. Biochem. Biophys. Res. Commun., 342: 1297-1304.
Rhee, J.S., S. Raisuddin, D.S. Hwang, K.W. Lee, I.C. Kim and J.S. Lee. 2009. Differential expression of metallothionein (MT) gene by trace metals and endocrinedisrupting chemicals in the hermaphroditic mangrove killifish, Kryptolebias marmoratus. Ecotoxicol. Environ. Saf., 72: 206-212.
Ringwood, A.H., M.J. Hameedi, R.F. Lee, M. Brower, E.C. Peters, G.I. Scott, S.N. Luoma and R.T. Di Giulio. 1999. Bivalve biomarker workshop: overview and discussion group summaries. Biomarkers, 4: 391-399.
Rodriguez-Ortega, M.J., A. Rodriguez-Ariza, J.L. Gomez- Ariza, A. Munoz-Serrano and J. Lopez-Barea. 2009. Multivariate discriminant analysis distinguishes metalfrom non metal-related biomarker responses in the clam Chamaelea gallina. Mar. Pollut. Bull., 58: 64-71.
Son, Y.-M. and H.-K. Byeon. 2005. The ichthyofauna and dynamics of the fish community in Miho stream, Korea. Kor. J. Ichthyol., 17: 271-278 (in Korean).
Song, H.Y., W.J. Kim, W.O. Lee and I.C. Bang. 2008. Morphological development of egg and larvae of Iksookimia choii (Cobitidae). Korean J. Limnol., 41: 104-110.
Swindell, W.R. 2011. Metallothionein and the biology of aging. Ageing Res. Rev., 10: 132-145.
Thirumoorthy, N., K.T.M. Kumar, A.S. Sundar, L. Panayappan and M. Chatterjee. 2007. Metallothionein: an overview, World J. Gastroenterol., 13: 993-996.
Wang, L., L. Song, D. Ni, H. Zhang and W. Liu. 2009. Alteration of metallothionein mRNA in bay scallop Argopecten irradians under cadmium exposure and bacteria challenge. Comp. Biochem. Physiol. C Pharmacol. Toxicol. Endocrinol., 149: 50-57.
Woo, S., S. Yum, J.H. Jung, W.J. Shim, C.H. Lee and T.K. Lee. 2006. Heavy metal-induced differential gene expression of metallothionein in Javanese medaka, Oryzias javanicus. Mar. Biotechnol., 8: 654-662.
Yan, C.H. and K.M. Chan. 2004. Cloning of zebrafish metallothionein gene and characterization of its gene promoter region in HepG2 cell line. Biochim. Biophys. Acta., 1679: 47-58.
※ AI-Helper는 부적절한 답변을 할 수 있습니다.