Cristina Sollars

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 γ-MSH sequence that is flanked by sets of paired basic amino acid proteolytic cleavage sites. The γ-MSH region in ray-finned fish POMCs either has degenerate cleavage sites or is totally absent in some species. However, the African lungfish γ-MSH sequence does contain a deletion which has not been observed in tetrapod γ-MSH sequences. The β-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 β-endorphin – features found in fish β-endorphin, but not tetrapod β-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 β-MSH sequence. Thus, like rat POMC, it is doubtful that β-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.

Cloning of a neoteleost (Oreochromis mossambicus) pro-opiomelanocortin (POMC) cDNA reveals a deletion of the γ-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.

Polygenic expression of somatostatin in the sturgeon Acipenser transmontanus: molecular cloning and distribution of the mRNAs encoding two somatostatin precursors.

The sequence of somatostatin‐14 (SS1) has been strongly preserved throughout the evolution of vertebrates from agnathans to mammals. In Acipenseridae (sturgeons), two isoforms of somatostatin have been characterized to date: somatostatin‐14 has been identified from the gastrointestinal tract of the pallid sturgeon Scaphirhynchus albus and [Pro2]somatostatin‐14 has been identified from the pituitary of the Russian sturgeon Acipenser gueldenstaedti. In the present study, we report the cloning of two distinct somatostatin cDNAs from the brain of the sturgeon Acipenser transmontanus. One of the cDNAs encodes a 116‐amino acid protein (PSS1) that contains the SS1 sequence at its C‐terminal extremity and, thus, is clearly orthologous to other vertebrate PSS1. The other cDNA encodes a 111‐amino acid protein that contains the somatostatin variant [Pro2]somatostatin‐14 at its C‐terminal extremity. This second precursor exhibits more than 67% identity with the recently characterized lungfish PSS2 and goldfish PSS2. Reverse transcriptase‐polymerase chain reaction analysis revealed that PSS1 is expressed in the central nervous system, the pancreas and the gut, whereas PSS2 is found in the central nervous system but not in the digestive system. In situ hybridization histochemistry showed that the PSS1 and PSS2 genes are differently expressed in numerous regions of the sturgeon brain. Interestingly, PSS1 and PSS2 mRNAs are present in the hypothalamus suggesting that, in sturgeon, both SS1 and SS2 may play hypophysiotropic functions. The PSS2 mRNA but not the PSS1 mRNA was found in the intermediate lobe of the pituitary. The present data demonstrate that two somatostatin genes are expressed in the sturgeon brain: one precursor generates somatostatin‐14 and the other one gives rise to a [Pro2]somatostatin‐14 variant, which is orthologous to goldfish, lungfish, and frog SS2. J. Comp. Neurol. 443:332–345, 2002.

Evaluating the radiation of the POMC gene in teleosts: characterization of American eel POMC

A distinctive feature of the pituitary hormone precursor, proopiomelanocortin (POMC), is the presence of multiple melanocortin core sequences (HFRW), and one copy of the opioid, beta-endorphin. In the older lineages of ray-finned fish (i.e., orders Acipenseriformes and Semionotiformes), certain extant lobe-finned fish (Australian lungfish and African lungfish), and the tetrapods there are three melanocortin regions in POMC: ACTH/alphaMSH, beta-MSH, and gamma-MSH. However, among the teleosts, the most recent radiation of the ray-finned fishes, the gamma-MSH sequence is absent from the POMC genes of euteleosts like the carp, tilapia, chum salmon, sockeye salmon, and rainbow trout. The objective of this study was to determine whether the gamma-MSH sequence still may be present in the POMC gene of a more basal lineage of the teleosts such as a representative from subdivision Elopomorpha. To this end, a POMC cDNA was cloned and sequenced from the pituitary of the American eel, Anguilla rostrata (order Anguilliformes, family Anguillidae). The open reading frame of the eel POMC cDNA was 648 nucleotides in length and encoded 216 amino acids. As predicted, eel POMC contained the deduced amino acid sequences for beta-endorphin, ACTH/alpha-MSH, and beta-MSH. These end-products displayed primary sequence features that are common to ray-finned fish. Eel POMC lacks a gamma-MSH sequence and a large portion of the joining peptide region. In this regard, the eel POMC gene thus displays features very similar to the POMC genes that have been sequenced from euteleosts. Although it is conceivable that the gamma-MSH sequence may be present in representatives from the other basal extant lineages of teleosts (i.e., subdivisions Osteoglossomorpha or Clupeomorpha), it is also possible that the deletion that resulted in the loss of the gamma-MSH sequence occurred in the ancestral neopterygian that gave rise to the teleosts. In this case, the gamma-MSH sequence should be absent in all extant teleosts.

Deciphering the origin of Met-enkephalin and Leu-enkephalin in Lobe-finned fish: cloning of australian lungfish proenkephalin.

The previous detection of Met-enkephalin and Leu-enkephalin in the CNS of the Australian lungfish, Neoceratodus forsteri, in a molar ratio comparable to mammals suggested that the lungfish proenkephalin precursor should contain the sequences of both Met-enkephalin and Leu-enkephalin as seen for mammalian proenkephalin. However, the cloning of a full-length proenkephalin cDNA from the CNS of the Australian lungfish indicates that the organization of this precursor is more similar to amphibian proenkephalin than mammalian proenkephalin. The Australian lungfish cDNA is 1284 nucleotides in length and the open reading frame (267 amino acids) contains seven opioid sequences (GenBank #AF232671). There are five copies of the Met-enkephalin sequence flanked by sets of paired basic amino acid proteolytic cleavage sites and two C-terminally extended forms of Met-enkephalin: YGGFMRSL and YGGFMGY. As seen for amphibians, no Leu-enkephalin sequence was detected in the Australian lungfish proenkephalin cDNA. The fact that Leu-enkephalin has been identified by radioimmunoassay and HPLC analysis in the CNS of the Australian lungfish indicates that a Leu-enkephalin-coding gene, distinct from proenkephalin, must be expressed in lungfish. Potential candidates may include a prodynorphin- or other opioid-like gene. Furthermore, the absence of a Leu-enkephalin sequence in lungfish and amphibian proenkephalin would suggest that the mutations that yielded this opioid sequence in tetrapod proenkephalin occurred at some point in the radiation of the amniote vertebrates.

Cloning of Prodynorphin cDNAs from the Brain of Australian and African Lungfish: Implications for the Evolution of the Prodynorphin Gene

In mammals the opioids Met-enkephalin and Leu-enkephalin are derived from a common precursor, proenkephalin, and as a result these neuropeptides are co-localized in enkephalinergic neurons. The mammalian scheme for enkephalinergic networks is not universal for all classes of sarcopterygian vertebrates. In an earlier study, distinct Met- and Leu-enkephalin-positive neurons were detected in the central nervous system (CNS) of the African lungfish, Protopterus annectens. More recently, characterization of proenkephalin cDNAs separately cloned from the CNS of P. annectens and the Australian lungfish, Neoceratodus forsteri, revealed that the proenkephalin gene in these species encodes only Met-enkephalin-related opioids. In the current study a full-length prodynorphin cDNA (accession No. AY 445637) was cloned and sequenced from the CNS of N. forsteri. In addition to encoding α-neoendorphin, dynorphin A and dynorphin B sequences unique to the lungfish, two Leu-enkephalin sequences, flanked by paired basic amino acid proteolytic cleavage sites, were detected in this precursor. The partial sequence of a P. annectens prodynorphin cDNA (accession No. AY445638) also encoded a Leu-enkephalin sequence and a novel YGGFF sequence. The presence of the Leu-enkephalin sequence in the lungfish prodynorphin precursors would explain the origin of the distinct Leu-enkephalin-positive neurons found in the African lungfish CNS. The realization that Met-enkephalin and Leu-enkephalin can be derived from distinct opioid-coding precursor genes calls into question the interpretation of comparative immunohistochemical studies that have mapped ‘enkephalinergic’ networks in non-mammalian vertebrates.

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.

Analyzing the radiation of the melanocortins in amphibians: cloning of POMC cDNAs from the pituitary of the urodele amphibians, Amphiuma means and Necturus maculosus.

Proopiomelanocortin (POMC) cDNAs were cloned and sequenced from brain extracts of two species of urodele amphibians: Amphiuma means and Necturus maculosus. Although the two species of urodele amphibians belong to separate families, and do not share a direct common ancestor, the level of primary sequence identity for the open reading of the POMC cDNAs was 90% at the amino acid level and 79% at the nucleotide level. It appears that the POMC gene in these urodele amphibians has been accumulating mutations at the amino acid level at a slower rate than the POMC gene in other sarcopterygian orders.