Cloning of proopiomelanocortin from the brain of the African lungfish, Protopterus annectens, and the brain of the western spadefoot toad, Spea multiplicatus

A degenerate primer, specific for the opioid core sequence YGGFM, was used to clone and sequence proopiomelanocortin (POMC) cDNAs from the brain of th e African lungfish, Protopterus annectens, and from the brain of the wester n spadefoot toad, Spea multiplicatus. In addition, the opioid-specific prim er was used to clone and sequence a 3'RACE product corresponding to a porti on of the open reading frame of S. multiplicatus proenkephalin. For both sp ecies, cDNA was made from a single brain and a degenerate opioid-specific p rimer provided a reliable probe for detecting opioid-related cDNAs. The Afr ican lungfish POMC cDNA was 1,168 nucleotides in length, and contained regi ons that are similar to tetrapod POMCs and fish POMCs. The African lungfish POMC encodes a tetrapod-like gamma-MSH sequence that is flanked by sets of paired basic amino acid proteolytic cleavage sites. The gamma-MSH region i n ray-finned fish POMCs either has degenerate cleavage sites or is totally absent in some species. However, the African lungfish gamma-MSH sequence do es contain a deletion which has not been observed in tetrapod gamma-MSH seq uences. The beta- endorphin region of lungfish POMC has the di-amino acid s equence tryptophan-aspartic acid in the N-terminal region and an additional glutamic acid residue in the C-terminal region of beta-endorphin - feature s found in fish beta-endorphin, but not tetrapod beta-endorphins. The weste rn spadefoot toad POMC was 1,186 nucleotides in length, and exhibited an or ganizational scheme typical for tetrapod POMCs. However, the toad POMC did lack a paired basic amino acid proteolytic cleavage site N-terminal to the beta-MSH sequence. Thus, like rat POMC, it is doubtful that beta-MSH is an end product in either the toad brain or intermediate pituitary. At the amin o acid level, the toad POMC had 76% sequence identity with Xenopus laevis P OMC and 68% sequence identity with Rana ribidunda POMC. The use of these PO MC sequences to assess phylogenetic relationships within anuran amphibians will be discussed. With respect to the fragment of S. multiplicatus proenke phalin cDNA, two metenkephalin sequences and the metenkephalin-Rf sequence were found encoded in this fragment. As seen for X. laevis and R. ridibunda proenkephalin, proenkephalin sequence was not detected in the C-terminal r egion of the S. multiplicatus proenkephalin. The absence of a leuenkephalin sequence may be a common feature of anuran amphibian proenkephalins.