Organization and promoter analysis of the grouper (Epinephelus coioides) epinecidin-1 gene.
Epinecidin-1, an antimicrobial peptide documented in some fish, is an essential component of the innate immune response in fish, but little is known about its gene regulation. To better understand the molecular mechanism controlling transcription of the epinecidin-1 gene, we cloned and sequenced the genes and promoter regions of three epinecidin-1 peptides from the grouper (Epinephelus coioides). These genes have the potential to encode three putative epinecidin-1 peptides with either a short or a long 5'-untranslated region (UTR). These epinecidin-1 genes, numbered 124-1 (gene structure: 5 exons), 124-2 (gene structure: 5 exons), and 961 (gene structure: 4 exons), have 3' UTR sequences that dramatically differ by being located on different exons in clones 124 and 961. To address the roles of lipopolysaccharide (LPS) and poly(I):poly(C) in regulating epinecidin-1 expression, serial deletions were prepared in the promoter region of two clones that contained three genes. Different fragments of the epinecidin-1 5'-flanking region were transfected into U937 (human histiocytic lymphoma) and ZFL (zebrafish liver) cells and then treated with 0, 1, 10, and 100 mug/mL LPS or poly(I):poly(C). The results showed that after treatment with 10 mug/mL LPS, high promoter activity was observed in the 0.6-kb promoter fragment (of clone 961). Promoter deletions showed that hepatocyte nuclear factor (HNF)-1 was required for a maximal response of epinecidin-1 961 promoter activity after LPS treatment in ZFL cells. Morphological studies of transgenic zebrafish indicated that the 2-kb epinecidin-1 124-1 promoter-driven GFP transcripts appeared in the eye and skin as confirmed by immunohistochemical staining. These results indicate that the 2-kb epinecidin-1 124-1 promoter is active in a tissue-specific manner.
Department of Environmental Biology and Fisheries Science, National Taiwan Ocean University, Keelung 202, Taiwan.
Chia-Yu
CY
Pan
Jyh-Yih
JY
Chen
I-Hsun
IH
Ni
Jen-Leih
JL
Wu
Ching-Ming
CM
Kuo
D020413:3' Untranslated Regions / Q000235:genetics; D020121:5' Untranslated Regions / Q000235:genetics; D000818:Animals; D030801:Animals, Genetically Modified; D023181:Antimicrobial Cationic Peptides / Q000235:genetics*; D001483:Base Sequence; D001492:Bass / Q000235:genetics*; D002478:Cells, Cultured; D004423:Ear; D029941:Fish Proteins / Q000235:genetics*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D051537:Hepatocyte Nuclear Factor 1 / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D008070:Lipopolysaccharides / Q000494:pharmacology; D008099:Liver / Q000166:cytology; D008969:Molecular Sequence Data; D011070:Poly I-C / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017422:Sequence Analysis, DNA; D012867:Skin / Q000378:metabolism; D014162:Transfection; D020298:U937 Cells; D015027:Zebrafish
D020413:3' Untranslated Regions; D020121:5' Untranslated Regions; D023181:Antimicrobial Cationic Peptides; D029941:Fish Proteins; D008070:Lipopolysaccharides; C521392:epinecidin-1, Epinephelus coioides; D051537:Hepatocyte Nuclear Factor 1; D049452:Green Fluorescent Proteins; D011070:Poly I-C