Molecular cloning and expression analysis of the ASC gene from mandarin fish and its regulation of NF-kappaB activation.
Apoptosis-associated speck-like protein containing a CARD (ASC) is an adaptor protein that has a bipartite domain structure, an N-terminal PYRIN domain and a C-terminal caspase-recruitment domain (CARD). In this study, we cloned the mandarin fish ASC cDNA (mfASC), which consisted of 899bp with a 115bp 5'-UTR and a 181bp 3'-UTR. The open reading frame encoded 201 amino acids. The mfASC shows 37% identity to an ASC orthologue from zebrafish. The mfASC has two protein-protein interaction domains, an N-terminal PYRIN domain and a C-terminal CARD domain. The mfASC gene structure was determined and had a length of 3954bp with four exons separated by three introns. Northern blot analysis showed that mfASC mRNA is constitutively expressed in the head kidney, gill, hind kidney, spleen and intestine. In vitro studies, mfASC fused with green fluorescent protein appeared as a speck in the transfected 293T cells. When transiently overexpressed in 293T cells, mfASC inhibited NF-kappaB activity with or without tumor necrosis factor (TNFalpha) or lipopolysacharide (LPS) stimulation.
Sun
Yanan
Y
State Key Laboratory for Biocontrol, School of Life Sciences, Sun Yat-sen University, Guangzhou 510275, PR China.
Wang
Jing
J
Lao
Haihua
H
Yin
Zhixin
Z
He
Wei
W
Weng
Shaoping
S
Yu
Xiaoqiang
X
Chan
SiuMing
S
He
Jianguo
J
D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D006801:Humans; D008070:Lipopolysaccharides / Q000276:immunology; D008969:Molecular Sequence Data; D016328:NF-kappa B / Q000378:metabolism*; D010473:Perciformes / Q000235:genetics* / Q000276:immunology* / Q000378:metabolism; D016415:Sequence Alignment; D014162:Transfection; D014409:Tumor Necrosis Factor-alpha / Q000276:immunology / Q000378:metabolism
D048868:Adaptor Proteins, Signal Transducing; D008070:Lipopolysaccharides; D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha