Metallothionein-2 gene from the mandarin fish Siniperca chuatsi: cDNA cloning, tissue expression, and immunohistochemical localization.
The metallothionein-2 (MT-2) gene was isolated from the mandarin fish, one of the most important industrial aquatic animals in China, by using rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of MT-2 comprised 60 amino acids and showed approximately 62.3% identity to human metallothionein. Its promoter region was amplified by thermal asymmetric interlaced polymerase chain reaction (TAIL-PCR). The MT-2 gene consists of 3 exons and 2 introns, extending approximately 900 bp of genomic sequence. Phylogenetic analysis clearly demonstrated that MT-2 formed a clade with fish metallothionein. The promoter region contained 5 putative metal-regulatory elements (MREs) and 1 TATA box. Real-time quantitative RT-PCR analysis revealed that MT-2 transcripts were significantly increased in the brain and gills and were stable in the muscles, liver, and trunk kidney in Cd(2+)-stimulated fish. Western blotting analysis demonstrated that the protein of the MT-2 gene was expressed mainly in the gills, liver, heart, trunk kidney, muscle, and intestine; it was weakly detected in the brain and head kidney. Moreover, the MT-2 protein was immunohistochemically detected in the cytoplasm in the liver and trunk kidney. All the above results revealed that the mandarin fish MT-2 would be a useful biomarker for metal pollution.
Gao
Dian
D
State Key Laboratory of Freshwater Ecology and Biotechnology, Chinese Academy of Sciences, Wuhan, Hubei Province, PR China.
Wang
Gui Tang
GT
Chen
Xing Tao
XT
Nie
Pin
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015415:Biomarkers / Q000378:metabolism; D015153:Blotting, Western; D019256:Cadmium Chloride / Q000633:toxicity; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004784:Environmental Monitoring / Q000379:methods; D005091:Exons; D029941:Fish Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000187:drug effects; D007150:Immunohistochemistry; D007438:Introns; D008099:Liver / Q000378:metabolism; D008668:Metallothionein / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D010473:Perciformes / Q000235:genetics* / Q000378:metabolism; D010802:Phylogeny; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014873:Water Pollutants / Q000633:toxicity
D015415:Biomarkers; D018076:DNA, Complementary; D029941:Fish Proteins; D012333:RNA, Messenger; D014873:Water Pollutants; D008668:Metallothionein; D019256:Cadmium Chloride