Sea bass ghrelin: molecular cloning and mRNA quantification during fasting and refeeding.
Ghrelin is a novel appetite-inducing peptide hormone secreted by the stomach. The purpose of this study was first to identify the cDNA encoding sequence for ghrelin in sea bass (Dicentrarchus labrax). Using molecular cloning techniques we sequenced the cDNA corresponding to sea bass ghrelin mRNA. A total of 798 bases including a 5'-untranslated region (89 bp), an open reading frame (ORF) (324 bp), and a 3'-untranslated region (385 bp) were detected. Nucleotide sequence (ORF) encoded a 108 amino acid prepropeptide that demonstrated complete conservation of the N-terminal "biological active core" (GSSF) of the predicted mature ghrelin peptide. We also analyzed fasting-induced changes in the expression of ghrelin mRNA, using a one-tube two-temperature real-time RT-PCR with which the gene expression can be absolutely quantified using the standard curve method. Our results revealed that ghrelin was highly expressed in the stomach with much lower levels of expression in the proximal intestine and brain. Levels of ghrelin mRNA in the stomach were upregulated under conditions of negative energy balance, such as starvation, and downregulated during positive energy balance, such as refeeding. These findings offer new information about the sea bass ghrelin gene and support a role of this orexigenic hormone in the regulation of food intake in sea bass.
Terova
Genciana
G
Department of Biotechnology and Molecular Sciences, University of Insubria, Via J.H.Dunant, 3- 21100 Varese, Italy. genciana.terova@uninsubria.it
Rimoldi
Simona
S
Bernardini
Giovanni
G
Gornati
Rosalba
R
Saroglia
Marco
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001492:Bass / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular / Q000379:methods; D018076:DNA, Complementary / Q000032:analysis; D004734:Energy Metabolism / Q000235:genetics / Q000502:physiology; D005215:Fasting / Q000378:metabolism*; D029941:Fish Proteins / Q000235:genetics* / Q000378:metabolism; D005786:Gene Expression Regulation / Q000502:physiology; D054439:Ghrelin / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009752:Nutritional Status / Q000235:genetics / Q000502:physiology; D012333:RNA, Messenger / Q000378:metabolism*; D016415:Sequence Alignment; D014018:Tissue Distribution
D018076:DNA, Complementary; D029941:Fish Proteins; D054439:Ghrelin; D012333:RNA, Messenger