cDNA cloning, pituitary location, and extra-pituitary expression of pro-opiomelanocortin gene in rare minnow (Gobiocypris rarus)

Abstract

A cDNA encoding pro-opiomelanocortin (POMC) gene was cloned from the pituitary gland of the rare minnow (Gobiocypris rarus), a small freshwater fish endemic to China. This was achieved by reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE). Data showed that the predicted rare minnow POMC (rmPOMC) cDNA consisted of 846bps coding for the following sequences, flanked by proteolytic cleavage sites: signal peptide (SP, Met(1)-Ala(28)), N-terminal peptide (Gln(29)-His(105)), ACTH (Ser(108)-Met(146)), α-MSH (Ser(108)-Gal(121)), CLIP (Pro(126)-Met(146)), β-LPH (Glu(149)-His(221)), γ-LPH (Glu1(49)-Ser(186)), β-MSH (Asp(170)-Ser(186)), and β-endorphin (β-EP, Tyr(189)-Gln(221)). Sequence analysis showed no region was homologous to γ-MSH (a tetrapod POMC feature). The amino acid sequence is highly similar to POMC-I and POMC-II of the common carp (92.4%), according to homologous alignment. It was POMCα through the phylogenetic analysis. Pituitary and extra-pituitary expression were studied using RT-PCR and in situ hybridization. The rmPOMC-positive cells were mainly located in the rostral pars distalis (RPD) and pars intermedia (PI). Some rmPOMC-positive cells were detected in the proximal pars distalis (PPD) as well, according to in situ hybridization. In the extra-pituitary tissues, positive signals were observed in the brain, intestines, gonads, hepatopancreas, spleen, and gills by RT-PCR analysis.