Organization of proenkephalin in amphibians: cloning of a proenkephalin cDNA from the brain of the anuran amphibian, Spea multiplicatus.

Cloning of a proenkephalin cDNA from the pelobatid anuran amphibian, Spea multiplicatus, provides additional evidence that Leu-enkephalin, although present in the brain of anuran amphibians, is not encoded by the proenkephalin gene. The S. multiplicatus proenkephalin cDNA is 1375 nucleotides in length, and the open reading frame contains the sequences of seven opioid sequences. There are five copies of the Met-enkephalin sequence, as well as an octapeptide opioid sequence (YGGFMRNY) and a heptapeptide opioid sequence (YGGFMRF). In the proenkephalin sequence of S. multiplicatus the penultimate opioid is a Met-enkephalin sequence rather than the Leu-enkephalin present in mammalian sequences. The same order of opioid sequences also is observed for the proenkephalin sequence of the pipid anuran amphibian, Xenopus laevis. Hence, from a phylogenetic standpoint the organization of tetrapod proenkephalin has been remarkably conserved. What remains to be resolved is whether the Leu-enkephalin sequence found in mammalian proenkephalin is an ancestral trait or a derived trait for the tetrapods. Unlike the proenkephalin precursor of X. laevis, all of the opioid sequences in the S. multiplicatus proenkephalin cDNA are flanked by paired-basic amino acid proteolytic cleavage sites. In this regard the proenkephalin sequence for S. multiplicatus is more similar to mammalian proenkephalins than the proenkephalin sequence of X. laevis. However, a comparison of the proenkephalin sequences in human, X. laevis, and S. multiplicatus revealed several conserved features in the evolution of the tetrapod proenkephalin gene. By contrast, a comparison of tetrapod proenkephalin sequences with the partial sequence of a sturgeon proenkephalin cDNA indicates that the position occupied by the penultimate opioid sequence in vertebrate proenkephalins may be a highly variable locus in this gene.

Cloning of a proopiomelanocortin cDNA from the pituitary of the Australian lungfish, Neoceratodus forsteri: analyzing trends in the organization of this prohormone precursor.

The polypeptide hormone precursor, proopiomelanocortin (POMC), was cloned and sequenced from the pituitary of the Australian lungfish, Neoceratodus forsteri, the only surviving species of the oldest extant lineage of lungfish. The Australian lungfish POMC cDNA had an open reading frame that coded for a 255-amino acid precursor. A comparison of POMC sequences from the Australian lungfish and the African lungfish indicated that the deduced amino acid sequences for ACTH, beta-MSH, and beta-endorphin were over 90% identical. Furthermore, within the open reading frames of the two lungfish POMCs, there was 84% identity at the nucleotide level. Although a gamma-MSH-like region was detected in the Australian lungfish POMC cDNA, this sequence contained mutations that have been detected in the gamma-MSH sequences of some ray-finned fish and are not found in the gamma-MSH sequence of the African lungfish or those of tetrapods. In addition, the sequence of beta-endorphin in the two species of lungfish has amino acid motifs that are found in the beta-endorphin sequences of cartilaginous fish and ray-finned fish but not in tetrapods. However, maximum parsimony analysis of the entire POMC open reading indicated that the lungfish POMC sequences form a clade with two amphibian POMC sequences rather than with POMC sequences from ray-finned fish. This result is consistent with the accepted view that the sarcopterygians (lungfishes and tetrapods) are a monophyletic assemblage. Analysis of rates of divergence for various POMC sequences indicate that point mutations are accumulating in the lungfish POMC sequences at a slower rate than in either amphibian or mammalian POMC sequences. The phylogenetic implications of these observations are discussed.

Duplication of the POMC gene in the paddlefish (Polyodon spathula): analysis of gamma-MSH, ACTH, and beta-endorphin regions of ray-finned fish POMC.

The proopiomelanocortin (POMC) gene, which encodes the common precursor for MSH-related and beta-endorphin-related end products, appeared early in chordate evolution and features a variety of lineage-specific modifications. Key among these has been the apparent degeneration and subsequent deletion of the gamma-MSH region during the evolution of POMC in the ray-finned fish. A second area of increasing focus has been the role of gene duplication in the evolution of POMC in particular and the opioid/orphanin gene family in general. The cloning and phylogenetic analysis of two POMC cDNAs from the paddlefish (Polyodon spathula) is reported here and biochemical data on their processed end products are presented. Based on conceptual amino acid translations, the paddlefish cDNAs encode all functional domains and, in most cases, the flanking paired-basic amino acid cleavage sites characteristic of gnathostome POMCs (i.e., signal sequence, gamma-MSH-like region, ACTH (alpha-MSH and CLIP), gamma-LPH, beta-MSH, and beta-endorphin). Phylogenetic analysis of the paddlefish POMC sequences in the context of the duplicated POMCs of sturgeon and salmonids suggests that degeneration of the gamma-MSH core sequence and its amino-terminal proteolytic cleavage site was initiated prior to divergence of the sturgeon and paddlefish lineages over 150 mya. Finally, a comparison of the relative rates of evolutionary divergence between paralogously related POMC genes within chondrostean and salmonid lineages provides potential support for the hypothesis that some taxa (e.g., the Chondrosteii) represent relic species as a result of an exceptionally slow rate of evolutionary change.

Cloning of a second proopiomelanocortin cDNA from the pituitary of the sturgeon, Acipenser transmontanus.

A recent study on the pituitary of the sturgeon, Acipenser transmontanus, resulted in the cloning of a cDNA that codes for the prohormone, proopiomelanocortin (POMC). This cDNA is designated sturgeon POMC A. Subsequent analysis of the sturgeon pituitary uncovered a second distinct POMC cDNA (sturgeon POMC B). In both sturgeon POMC cDNAs the open reading frame is 795 nucleotides in length. However, the two sturgeon POMC cDNAs differ at 26 amino acid positions in the opening frame. In addition, the 2 forms of POMC differ at 45 nucleotide positions within the open reading frame. The number and types of point mutations are compared in the 2 sturgeons POMC cDNAs, and the origin of the two POMC genes is discussed.

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 the African lungfish, Protopterus annectens, and from the brain of the western spadefoot toad, Spea multiplicatus. In addition, the opioid-specific primer was used to clone and sequence a 3'RACE product corresponding to a portion of the open reading frame of S. multiplicatus proenkephalin. For both species, cDNA was made from a single brain and a degenerate opioid-specific primer provided a reliable probe for detecting opioid-related cDNAs. The African lungfish POMC cDNA was 1,168 nucleotides in length, and contained regions 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 in ray-finned fish POMCs either has degenerate cleavage sites or is totally absent in some species. However, the African lungfish gamma-MSH sequence does contain a deletion which has not been observed in tetrapod gamma-MSH sequences. The beta-endorphin region of lungfish POMC has the di-amino acid sequence tryptophan-aspartic acid in the N-terminal region and an additional glutamic acid residue in the C-terminal region of beta-endorphin - features found in fish beta-endorphin, but not tetrapod beta-endorphins. The western spadefoot toad POMC was 1,186 nucleotides in length, and exhibited an organizational 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 amino acid level, the toad POMC had 76% sequence identity with Xenopus laevis POMC and 68% sequence identity with Rana ribidunda POMC. The use of these POMC sequences to assess phylogenetic relationships within anuran amphibians will be discussed. With respect to the fragment of S. multiplicatus proenkephalin cDNA, two metenkephalin sequences and the metenkephalin-RF sequence were found encoded in this fragment. As seen for X. laevis and R. ridibunda proenkephalin, a leuenkephalin sequence was not detected in the C-terminal region of the S. multiplicatus proenkephalin. The absence of a leuenkephalin sequence may be a common feature of anuran amphibian proenkephalins.

Studies on the GH/SL gene family: cloning of African lungfish (Protopterus annectens) growth hormone and somatolactin and toad (Bufo marinus) growth hormone.

The lungfishes (lobe-finned fish) occupy a unique position in vertebrate phylogeny, being regarded as the closest extant relatives to the tetrapods. The putative pituitary hormone somatolactin (SL) has hitherto been found only in teleost fishes, and the presence of this protein in tetrapods or lobe-finned fishes has not been ascertained. It was therefore of interest to determine the structure of SL in the African lungfish (Protopterus annectens), as this information would be useful for designing probes to facilitate the detection of SL genes in amphibians and other tetrapods. The structural relationships between SL, growth hormone (GH), and prolactin (PRL) strongly suggest that these proteins evolved from a common ancestor. To obtain a more complete picture of the evolution of these hormones in lungfish, African lungfish GH has been cloned and sequenced. The cDNA sequence of a toad (Bufo marinus) GH was determined to facilitate maximum parsimony analysis of GH sequences. Cladistic analysis confirmed that lungfish and amphibian GH sequences form a clade distinct from the GH sequences of ray-finned fishes. A distance matrix analysis of SL sequences indicated that lungfish SL had the lowest primary sequence identity with goldfish SL (47%) and the highest with flounder SL (66%). The detection of SL in a lungfish indicates that the gene duplication within the SL/GH/PRL family, which gave rise to SL, must have occurred in a common ancestor of the ray-finned fishes (Actinopterygii) and the lungfishes (Sarcopterygii) and tetrapods.

Cloning of a neoteleost (Oreochromis mossambicus) pro-opiomelanocortin (POMC) cDNA reveals a deletion of the gamma-melanotropin region and most of the joining peptide region: implications for POMC processing.

A signature feature of tetrapod pro-opiomelanocortin (POMC) is the presence of three melantropin (MSH) coding regions (alpha-MSH, beta-MSH, gamma-MSH). The MSH duplication events occurred early during the radiation of the jawed vertebrates well over 400 million years ago. However, in at least one order of modern bony fish (subdivision Teleostei; order Salmoniformes; i.e. salmon and trout) the gamma-MSH sequence has been deleted from POMC. To determine whether the gamma-MSH deletion has occurred in other teleost orders, a POMC cDNA was cloned from the pituitary of the neoteleost Oreochromis mossambicus (order Perciformes). In O. mossambicus POMC, the deletion is more extensive and includes the gamma-MSH sequence and most of the joining peptide region. Because the salmoniform and perciform teleosts do not share a direct common ancestor, the gamma-MSH deletion event must have occurred early in the evolution of the neoteleost fishes. The post-translational processing of O. mossambicus POMC occurs despite the fact that the proteolytic recognition sequence, (R/K)-Xn-(R/K) where n can be 0, 2, 4, or 6, a common feature in mammalian neuropeptide and polypeptide hormone precursors, is not present at several cleavage sites in O. mossambicus POMC. These observations would indicate that either the prohormone convertases in teleost fish use distinct recognition sequences or vertebrate prohormone convertases are capable of recognizing a greater number of primary sequence motifs around proteolytic cleavage sites.