Tracking the evolution of the proenkephalin gene in tetrapods

In gnathostomes there is remarkable consistency in the organization of the proenkephalin gene. This opioid precursor encodes seven opioid (YGGF) sequences: five pentapeptide sequences, a met-enkephalin-7 sequence and a met-enkephalin-8 sequence. Yet, within vertebrate lineages there can be distinct sets of pentapeptide opioids (YGGFM or YGGFL). In the Sarcopterygii, the sixth opioid position in lungfishes and anuran amphibian proenkephalin genes encodes a met-enkephalin (YGGFM) sequence. However, in mammalian proenkephalin there is a leu-enkephalin (YGGFL) sequence at this position. This study was done to test the hypothesis that the presence of the leu-enkephalin sequence in mammals is a feature common to amniote vertebrates, but not present in anamniote vertebrates. To resolve this issue, proenkephalin cDNAs were cloned from the urodele amphibians, Amphiuma means and Necturus maculosus, and two amniote vertebrates, the turtle, Chrysemys scripta, and the brown snake, Storeria dekayi. As predicted, a met-enkephalin sequence is present at the sixth opioid position in urodele amphibians; whereas, a leu-enkephalin sequence is present at this opioid site in the reptile proenkephalin sequences. These data are consistent with the conclusion that the transition from a met-enkephalin sequence to a leu-enkephalin sequence at the sixth opioid position in tetrapod proenkephalins occurred in the ancestral proto-reptiles. Phylogenetic analyses, using the Maximum Parsimony and Neighbor-Joining algorithms, of the amphibian proenkephalin sequences supported the position that anuran and urodele amphibians are a monophyletic assemblage. The same analysis of reptile-related proenkephalin sequences, including the deduced amino acid sequence of a partially characterized alligator proenkephalin cDNA, could not conclusively resolve the phylogeny of the major reptilian orders.