Proopiomelanocortin cDNA Research Articles

Cloning of POMC cDNA and the stress response in Coilia nasus

PDF HTML阅读 XML下载 导出引用 引用提醒 刀鲚POMC基因的cDNA克隆及其应激应答 DOI: 作者: 作者单位: 1. 农业部淡水渔业健康养殖重点实验室, 浙江省淡水水产遗传育种重点实验室, 浙江省淡水水产研究所, 浙江 湖州 313001;2. 中国水产科学研究院 淡水渔业研究中心, 农业部淡水渔业和种质资源利用重点实验室, 江苏 无锡 214081 作者简介: 杜富宽(1983-),男,助理研究员.E-mail:adublg@126.com 通讯作者: 中图分类号: S96 基金项目: 农业部淡水渔业健康养殖重点实验室开放课题（ZJK2014003）；中国水产科学研究院淡水渔业研究中心中央级公益性科研院所基本科研业务费专项资金（2015JBFR06）；江苏省科技支撑项目（BE2014307）. Cloning of POMC cDNA and the stress response in Coilia nasus Author: Affiliation: 1. Key Laboratory of Healthy and Freshwater Aquaculture, Ministry of Agriculture;Key Laboratory of Freshwater Aquaculture Genetic and Breeding of Zhejiang Province, Zhejiang Institute of Freshwater Fisheries, Huzhou 313001, China;2. Key Laboratory of Freshwater Fisheries and Germplasm Resources Utilization, Ministry of Agriculture;Freshwater Fisheries Research Center, Chinese Academy of Fishery Sciences, Wuxi 214081, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为了研究刀鲚（POMC基因的cDNA全长。刀鲚基因的cDNA全长2030 bp，编码区699 bp，编码232个氨基酸。二级结构预测显示，刀鲚POMC包括信号肽（signal peptide）、N末端区（N-terminal region，NPP）、促肾上腺皮质激素结构域（adrenocorticotropic hormone，ACTH）、γ-脂解素（-EP）结构域。运用荧光定量的方法检测了该基因在刀鲚不同组织中的表达分布。结果显示，基因的表达量显著下降（<0.05），6 h的表达量恢复至应激前。基因在脂肪代谢及应激调控中发挥重要的功能，本研究结果可为该基因在刀鲚后续育种及应激调控工作提供重要基础。 Abstract:The estuarine tapertail anchovy () is widely distributed in the Yangtze River, the coastal waters of China and Korea, and the Ariake Sound of Japan. It is a commercially important species because of its nutritive value and popularity. However, excess fishing and changes in aquatic ecology have almost caused extinction of this species in the middle reaches of the Yangtze River. In recent years, several studies have been performed on pond breeding and artificial propagation, and larval rearing techniques have been established. Thus, the threat to is very sensitive to stress, which often results in death and huge economic losses. Little is known about the mechanism by which loading stress causes death in fish. Pro-opiomelanocortin (POMC) is synthesized in the corticotrophs and melanotrophs of the anterior and intermediate lobes of the pituitary, respectively, as well as in peptidergic neurons in the arcuate nucleus of the hypothalamus. It is post-translationally cleaved into peptide hormones, including adrenocorticotrophic hormone, -melanocyte stimulating hormone (MSH), N-POMC, and -LPH) in tissue- and cell dependent manners. These peptides exhibit different physiological functions, such as promoting ovarian maturation, lipolysis, inhibiting fat accumulation, regulating osmotic pressure, and reacting to stressors. We cloned the and determined that the full-length cDNA is 1212 bp long, with a 699 bp open reading frame that encoded a 232 amino acid protein. The POMC protein is predicted to contain the typical POMC domains of a signal peptide sequence, the N-terminal region, and the adrenocorticotrophic hormone, -EP domains. Gene expression patterns were detected in different tissues by quantitative reverse transcription-polymerasechain reaction. was expressed strongly in the brain and relatively highly in the gill, kidney, and testis, whereas it was expressed at low levels in the liver, spleen, intestine, head kidney, muscle, and ovary. <0.05) and was significantly up regulated 4 h and 6 h after transport ( expression returned to the control level before stress. The gene plays an important role regulating fat metabolism and stress. Our results provide important clues for breeding and stress control studies in . 参考文献 相似文献 引证文献

60 YEARS OF POMC: The proopiomelanocortin gene: discovery, deletion and disease.

The cloning of the bovine proopiomelanocortin (POMC) cDNA in 1978 by Nakanishi and colleagues was the result of a remarkable series of exacting and ingenious experiments. With this work, they instantly confirmed the single precursor hypothesis for adrenocorticotrophic hormone-β-lipotropin, as it was then known, and in so doing revealed the existence of additional, largely unpredicted, N-terminal peptides that together formed the POMC precursor peptide. This work paved the way for a host of additional studies into the physiology of these peptides and their regulation. Furthermore, the cloning of the murine Pomc gene was essential for subsequent studies, in which Pomc was intentionally deleted in the mouse illuminating its substantial role in body weight regulation and adrenal function. Contemporaneously with this work, naturally occurring mutations in human POMC came to light underlining the vital role of this gene in appetite regulation. This article reviews each of these aspects of POMC with the benefit of several decades of hindsight and informed by more recent genomic and transcriptomic data.

Functional relevance of three proopiomelanocortin (POMC) genes in darkening camouflage, blind-side hypermelanosis, and appetite of Paralichthys olivaceus.

To determine whether proopiomelanocortin (POMC) genes are involved in darkening color camouflage, blind-side hypermelanosis, and appetite in flatfish, we isolated and cloned three POMC genes from the pituitary of the olive flounder (Paralichthys olivaceus) and compared their amino acid (aa) structures to those of POMC genes from other animals. Next, we examined the relationship of these pituitary POMC genes to camouflage color change, blind-side hypermelanosis, and appetite by quantifying mRNA expression. Olive flounder (of)-POMC1, 2, and 3 cDNAs consisted of 648-bp, 582-bp, and 693-bp open reading frames (ORF) encoding 216 aa, 194 aa, and 231 aa residues, respectively. Structurally, the three of-POMC cDNAs consisted of seven peptides (signal peptide, N-POMC, α-MSH, CLIP, N-β-LPH, β-MSH and β-END [or END-like peptide]) that are similar to those of other fish POMC cDNAs. α-MSH encoded a protein composed of 13 aa and β-MSH encoded a protein composed of 17 aa. The three POMC genes were predominantly expressed in the pituitary gland, but they were also expressed in a variety of tissues, including brain, eye, kidney, heart, testis, and skin. of-POMC2 exhibited the highest expression, while of-POMC3 displayed the lowest expression. The relative levels of of-POMC1 and 3 mRNAs were not influenced by background color and feeding (or fasting), but the relative level of of-POMC2 mRNA significantly increased in response to a dark background and fasting. The relative levels of of-POMC1 and 2 mRNAs were significantly higher in hypermelanic fish; however, we did not determine a direct anorexigenic or orexigenic relationship for the three POMC genes. These results indicate that pituitary POMC genes are related to darkening color change and the differentiation of pigment cells, but they are not directly related to appetite.

Molecular cloning of propiomelanocortin cDNA and multi-tissue mRNA expression in Parasilurus asotus

We investigated the structure,evolution,and tissue specific expression of Parasilurus asotus proopiomelanocortin(POMC) cDNA using RT-PCR and RACE.The POMC cDNA was 1 099 bp in length and contained a 129 bp 5′UTR,a 322 bp 3′UTR,and a 648 bp open reading frame encoding a protein of 215 amino acids.The catfish POMC protein contained a signal peptide(SP,Met1-Ala28),an N-terminal peptide(Gln29-Asp102),adrenocorticotropic hormone(ACTH,Ser105-Met144),α-melanocyte stimulating hormone(α-MSH,Ser105-Val117),corticotropin-like intermediate lobe peptide(CLIP,Arg123–Met144),β-lipotropin(β-LPH,Glu145-Ser158),β-MSH(Asp161-Ser177),and β-Endorphin(β-EP,Tyr178-Leu215).Catfish POMC protein does not contain a γ-MSH region and the majority of the joining peptide and part of the γ-LPH were deleted.The protein shared the highest similarity(88%) with channel catfish(Parasilurus asotus) POMC.The most highly conserved active peptide was α-MSH,followed by β-MSH,β-EP,and CLIP whereas γ-MSH,joining peptide(JP),and γ-LPH were located in the most divergent region.The protein had a calculated molecular weight of 24.66 kD,a theoreti-cal isoelectric point of 7.25,was hydrophilic in most regions,and was rich in B cell antigenicity positions.Catfish POMC mRNA was primarily expressed in the pituitary and the concentration of mRNA was much higher in brain than in other tissues.

Molecular cloning, tissue distribution and quantitative analysis of two proopiomelanocortin mRNAs in Japanese flounder (Paralichthys olivaceus)

Proopiomelanocortin (POMC) plays an essential role in the stress response of the hypothalamic-pituitary-adrenal axis, and is the precursor of biologically active peptides such as adrenocorticotropin (ACTH), alpha-melanocyte-stimulating hormone (alpha-MSH), beta-melanocyte-stimulation hormone (beta-MSH) and beta-endorphin. We have synthesized two different forms of POMC cDNA clones, POMC-I and POMC-II, from a pituitary cDNA library for Paralichthys olivaceus, or Japanese flounder. jfPOMC-I cDNA consists of 954bp and encodes a polypeptide of 216 amino acid residues, whereas jfPOMC-II consists of 971bp which encode a polypeptide of 194 amino acid residues. The high levels of jfPOMC-I and -II mRNAs detected in the pituitary tissue and moderate levels detected in the brain tissue plus our quantitative RT-PCR analysis, which showed there to be no significant difference between the levels of jfPOMC-I and -II mRNAs, indicate that there may be no functional separation between these two mRNAs in the flounder.

Cloning of sole proopiomelanocortin (POMC) cDNA and the effects of stocking density on POMC mRNA and growth rate in sole, Solea solea

Proopiomelanocortin (POMC) is an important gene implicated in different functions, such as the stress response of the hypothalamus–pituitary–adrenal axis. The aim of the present study was to determine whether farming conditions, such as stocking density, can be considered a powerful stressor influencing in turn the growth rate in juvenile fish. Thus, POMC cDNA expression was investigated during adaptation to farming conditions in sole ( Solea solea), as a model for studying the effects of rearing densities on stress response; different stocking densities (50, 100, and 250 animals/m 2) were applied and, after 7 and 21 days, the fishes were examined for body weight and plasma cortisol levels as indicators of stress. In addition, proopiomelanocortin was cloned and sequenced from the brain of sole, allowing semi-quantitative RT-PCR to be performed to evaluate POMC mRNA expression in brain tissue. There was a significant increase in cortisol levels in fish reared at high stocking densities of 250/m 2 compared to fish reared at control densities of 100 and 50/m 2, in both experimental times, i.e., 7 and 21 days. The high stocking densities were also found to decrease the specific growth rate of fish. Moreover, it was demonstrated that the highest stocking density induced a significant decrease in sole POMC mRNA expression. It is concluded that POMC and cortisol are both involved in the stress response due to high rearing densities, during which cortisol may serve as a negative regulator of POMC.

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.

Nucleotide sequence and expression of three subtypes of proopiomelanocortin mRNA in barfin flounder

Melanophore-stimulating hormone (MSH) has been shown to be associated with food intake in addition to body color change in teleosts. MSH is encoded by a proopiomelanocortin (POMC) gene together with endorphin (END). To assess the significance of MSH to biological activities, we determined the structure and evaluated the expression of POMC mRNA in barfin flounder (bf), Verasper moseri, a member of a group of teleosts, Pleuronectiformes. Three subtypes of POMC cDNAs (A, B, and C) were amplified from bf pituitary glands. These bfPOMCs contained segments for N-POMC, α-MSH, β-MSH, and β-END as do other teleost POMCs, while POMC-C showed remarkable variations in the segments corresponding to N-POMC and β-END. A phylogenetic tree of ray-finned fish POMCs constructed by the neighbor joining method revealed that the three POMC subtypes may have appeared as a result of duplication events occurring at least twice during the course of bf evolution. The first duplication may have generated the lineage leading to an ancestor of bfPOMC-A and -B and that leading to bfPOMC-C, and then the lineage of bfPOMC-A may have diverged from that of bfPOMC-B. All peptides flanked by processing signals excluding N-POMC-C (1–14) were identified in a single pituitary extract by mass spectrometry, and the cDNAs of three POMCs were amplified from a single pituitary by reverse transcription polymerase chain reaction. These results demonstrated that the three POMC genes are expressed in a single individual. While the bfPOMC-A gene was exclusively expressed in the pituitary, the bfPOMC-B and -C genes were expressed in non-pituitary tissues such as brain, gill, heart, spleen, liver, stomach, intestine, testis, muscle, blood, and skin in addition to the pituitary. The expression levels of the POMC-A, -B, and -C genes in pituitary neurointermediate lobe were greater in the fish reared with a black background than the fish reared with a white background, indicating that MSH derived from all of the three bfPOMC genes was associated with body color change. No difference was observed in the expression levels of bfPOMC-C in the brain in response to feeding status.

Molecular characterization of the leopard gecko POMC gene and expressional change in the testis by acclimation to low temperature and with a short photoperiod

The gene for proopiomelanocortin (POMC), a common precursor of malanotropins, corticotropin, and β-endorphin, was isolated and analyzed in the squamata species, the leopard gecko, Eublepharis macularius. Leopard gecko POMC (lgPOMC) cDNA is composed of 1299 bp, excluding the poly(A) tail, and encodes 270 amino acids. The deduced amino acid sequence showed the same structural organization as that of other species and displayed identity with those of other vertebrates: 68% with mud turtles, 57/57% with African clawed frog A/B, 53% with chickens, and 45% with mice. In a phylogenic tree, the lgPOMC clustered with the sequences of the mud turtle POMC and python POMC. The lgPOMC gene comprises three exons and two introns and this structure is consistent with humans, rats, mice, African clawed frog and zebrafish. RT-PCR analysis revealed that the lgPOMC mRNA was expressed only in the whole brain, pituitary, and gonads. To analyze in more detail, a competitive assay system to quantify the expression levels of POMC mRNA was established. We measured the POMC mRNA expression levels in the leopard gecko testes following transfer from a condition of 29 °C, 16L/8D to 18 °C, 10L/14D over 6 weeks. This 6-week acclimation increased the POMC mRNA expression levels significantly. This suggests that the leopard gecko POMC-derived peptides play a role in the mediation of the effect of environmental factors on reproduction.

Molecular cloning of proopiomelanocortin cDNA and multi-tissue mRNA expression in channel catfish

Channel catfish ( Ictalurus punctatus) proopiomelanocortin ( POMC) cDNA was cloned to investigate its structure, evolution, and expression in different tissues. POMC is an important gene in the hypothalamus–pituitary–adrenal axis, the main mediator of the stress response. POMC gene was isolated from a pituitary cDNA library and nucleotide sequence was determined. POMC cDNA is composed of 1164 nucleotides with a 639 nucleotide open reading frame encoding a protein of 212 amino acids. Catfish POMC protein contains a signal peptide (SP, Met 1–Ala 28), N-terminal peptide (Gln 29–Glu 101), adrenocorticotropic hormone (ACTH, Ser 104–Met 142), α-melanocyte stimulating hormone (α-MSH, Ser 104–Val 116), corticotropin-like intermediate lobe peptide (CLIP, Arg 121–Met 142), β-lipotropin (β-LPH, Glu 145–His 212), γ-lipotropin (γ-LPH, Glu 145– Ser 177), β-MSH (Asp 161–Ser 177), and β-endorphin (β-EP, Tyr 180–His 212). Catfish POMC protein does not contain a γ-MSH region and most of the joining peptide and part of the γ-LPH are deleted. Protein sequence alignment showed the highest similarity with the carp ( Cyprinus carpio) POMC I (66.5%) and POMC II (67%), while the sea lamprey ( Petromyzon marinus) POC (17.9%) and POM (18.8%) were the most divergent. The average similarity was 46.95% among the 44 POMC proteins from 36 different species analyzed. Compared to the POMC mRNA levels in the pituitary, the concentration of the POMC mRNA was 0.0594% in the anterior kidney and 0.0012–0.0045% in all the other tissues except in the skin where the lowest expression (0.0005%) was observed. Overall architecture of channel catfish POMC is highly similar to those from other teleosts.

Characterizing a proopiomelanocortin cDNA cloned from the brain of the Bichir, Polypterus senegalus: evaluating phylogenetic relationships among ray-finned fish

There is general agreement that the polypteriform fishes, like Polypterus senegalus, constitute a unique lineage in the evolution of the vertebrates. However, the precise position of these fishes had been a point of controversy since the time of Darwin and Huxley. There is now consensus that the polypteriform fishes are members of superorder Actinopterygii. However, within the Actinopterygii, it is still debatable as to whether the polypteriform fishes are an early offshoot of the Actinopterygii or a more recent sister group to the sturgeon and other extant chondrostean fishes. In this study the sequence of proopiomelanocortin (POMC), the common precursor for the melanocortins and β-endorphin, was used to evaluate the phylogenetic position of the polypteriform fishes relative to other bony fishes. 3 ′RACE and 5 ′RACE protocols were used to amplify overlapping regions of a POMC cDNA from the brain of P. senegalus. The full-length POMC cDNA had an open reading frame that encoded 259 amino acids. As seen in most gnathostomes, P. senegalus POMC has three melanocortin sequences (ACTH/α-MSH, γ-MSH, and β-MSH), and a β-endorphin region. For phylogenetic analysis, the following POMC sequences were aligned at the amino acid level and analyzed using a maximum parsimony algorithm: P. senegalus, dogfish, sturgeon A, paddlefish A, sockeye salmon A, tilapia, and gar. The dogfish POMC sequence was used as the out-group. In this analysis the P. senegalus POMC sequence formed a clade with the chondrostean POMC sequences (sturgeon A and paddlefish A), and not with the neopterygian sequences (sockeye salmon A, tilapia, and gar). P. senegalus POMC is remarkably similar to sturgeon POMC A. In particular, in both precursors there is evidence for degeneration at the proteolytic cleavage site that precedes the γ-MSH sequence. Based on the analysis of this nuclear gene it would appear that P. senegalus belongs to a branch of the chrondrostean lineage rather than representing a lineage of ray-finned fish that is ancestral to the chondrostean and neoptyergian ray-finned fishes. Alternatively, if the polypteriform fishes are in fact an early offshoot of the Actinopterygii (the traditional view), then the observations made for P. senegalus POMC relative to the chondrostean POMC sequences is the result of convergence.

Gene Therapy for Bladder Pain With Gene Gun Particle Encoding Pro-Opiomelanocortin cDNA

Interstitial cystitis is a bladder hypersensitivity disease associated with bladder pain that has been a major challenge to understand and treat. We hypothesized that targeted and localized expression of endogenous opioid peptide in the bladder could be useful for the treatment of bladder pain. Pro-opiomelanocortin (POMC) is one of such precursor molecules. In this study we developed a gene gun method for the transfer of POMC cDNA in vivo and investigated its therapeutic effect on acetic acid induced bladder hyperactivity in rats. Human POMC cDNA was cloned into a modified pCMV plasmid and delivered into the bladder wall of adult female rats by direct injection or the gene gun. Three days after gene therapy continuous cystometrograms were performed using urethane anesthesia by filling the bladder (0.08 ml per minute) with saline, followed by 0.3% acetic acid. Bladder immunohistochemical testing was used to detect endorphin after POMC cDNA transfer. The intercontraction interval was decreased after intravesical instillation of acetic acid (73.1% or 68.1% decrease) in 2 control groups treated with saline or the gene gun without POMC cDNA, respectively. However, rats that received POMC cDNA via the gene gun showed a significantly decreased response (intercontraction interval 35% decreased) to acetic acid instillation, whereas this antinociceptive effect was not detected in the plasmid POMC cDNA direct injection group. This effect induced by POMC gene gun treatment was reversed by intramuscular naloxone (1 mg/kg), an opioid antagonist. Increased endorphin immunoreactivity with anti-endorphin antibodies was observed in the bladder of gene gun treated animals. The POMC gene can be transferred in the bladder using the gene gun and increased bladder expression of endorphin can suppress nociceptive responses induced by bladder irritation. Thus, POMC gene gun delivery may be useful for the treatment of interstitial cystitis and other types of visceral pain.

Intramuscular electroporation with the pro-opiomelanocortin gene in rat adjuvant arthritis

Endogenous opioid peptides have an essential role in the intrinsic modulation and control of inflammatory pain, which could be therapeutically useful. In this study, we established a muscular electroporation method for the gene transfer of pro-opiomelanocortin (POMC) in vivo and investigated its effect on inflammatory pain in a rat model of rheumatoid arthritis. The gene encoding human POMC was inserted into a modified pCMV plasmid, and 0–200 μg of the plasmid-POMC DNA construct was transferred into the tibialis anterior muscle of rats treated with complete Freund's adjuvant (CFA) with or without POMC gene transfer by the electroporation method. The safety and efficiency of the gene transfer was assessed with the following parameters: thermal hyperalgesia, serum adrenocorticotropic hormone (ACTH) and endorphin levels, paw swelling and muscle endorphin levels at 1, 2 and 3 weeks after electroporation. Serum ACTH and endorphin levels of the group into which the gene encoding POMC had been transferred were increased to about 13–14-fold those of the normal control. These levels peaked 1 week after electroporation and significantly decreased 2 weeks after electroporation. Rats that had received the gene encoding POMC had less thermal hypersensitivity and paw swelling than the non-gene-transferred group at days 3, 5 and 7 after injection with CFA. Our promising results showed that transfer of the gene encoding POMC by electroporation is a new and effective method for its expression in vivo, and the analgesic effects of POMC cDNA with electroporation in a rat model of rheumatoid arthritis are reversed by naloxone.

Molecular cloning of proopiomelanocortin cDNA in the ratfish, a holocephalan

Proopiomelanocortin (POMC) is a precursor for several pituitary hormones including adrenocorticotropin (ACTH), melanocyte-stimulating hormone (MSH) and endorphin (END). Fish POMCs in four taxonomic classes, Cephalaspidomorphi (lampreys), Chondrichthyes (cartilaginous fish), Sarcopterygii (lobe-finned fish), and Actinopterygii (ray-finned fish) have been identified. However, two essential species, ratfish in Chondrichthyes and hagfish in Agnatha, are still missing in the evolutionary image of this molecule. The present study reports analysis of POMC cDNA in the ratfish, Chimaera phantasma, which belongs to another subclass in the Chondrichthyes. Partial cDNA clones were amplified by PCR from single-strand cDNA prepared on total RNA from a complex of pituitary and hypothalamus, and subsequently overlapped to obtain a full-length sequence. Ratfish POMC cDNA consists of 1294 bp excluding the poly(A) tail. It encodes a signal peptide of 25 amino acids and POMC of 300 amino acids. γ-MSH, ACTH, α-MSH, δ-MSH, β-MSH, and β-END are located at prePOMC (76–87), (120–158), (120–132), (212–227), (275–290), and (293–325), respectively. δ-MSH, originally found in elasmobranch POMCs, was also present in ratfish POMC, suggesting this structure might have appeared after the divergence of chondrichthians from the ancestral lineage. Thus, we demonstrated the common occurrence of four MSHs in chondrichthian POMC and established a clear understanding of the molecular evolution of POMC in gnathostomes.

Syrian hamster proopiomelanocortin cDNA cloning and early seasonal changes in testicular expression

Beta-endorphin (β-End) and its precursor, proopiomelanocortin (POMC), are expressed in testis and β-End stimulates testosterone secretion locally. We measured POMC expression in Syrian hamster testis following transfer from long days (LDs) to short days (SDs). We used RT-PCR to amplify partial-length hamster POMC cDNA to generate a probe for Northern analysis. We also used rat β-End antiserum for radioimmunoassay analysis. SD exposure for 2 weeks decreased POMC mRNA and β-End, but not testis weight or testosterone. We conclude that POMC signaling may play a role in seasonal regulation of testicular function.

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, β-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/αMSH, β-MSH, and γ-MSH. However, among the teleosts, the most recent radiation of the ray-finned fishes, the γ-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 γ-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 β-endorphin, ACTH/α-MSH, and β-MSH. These end-products displayed primary sequence features that are common to ray-finned fish. Eel POMC lacks a γ-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 γ-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 γ-MSH sequence occurred in the ancestral neopterygian that gave rise to the teleosts. In this case, the γ-MSH sequence should be absent in all extant teleosts.

Presence of the δ-MSH sequence in a proopiomelanocortin cDNA cloned from the pituitary of the galeoid shark, Heterodontus portusjacksoni

Since a fourth MSH sequence, δ-MSH, has been detected in the proopiomelanocortin (POMC) gene of a dogfish and a stingray, members of superorder Squalea (class Chondrichthyes), it is possible that this novel MSH sequence might be a feature common to the POMC genes of all modern sharks and rays. As an initial step towards addressing this question, a full-length POMC cDNA was cloned and sequenced from the pituitary of the Port Jackson shark, Heterodontus portusjacksoni. The Port Jackson shark represents one of the oldest lineages in superorder Galea, and this superorder together with superorder Squalea form infraclass Neoselachii (the extant sharks and rays). The Port Jackson shark POMC cDNA has an open reading frame that is 1032 nucleotides in length and encodes the deduced amino acids sequences for β-endorphin, ACTH/α-MSH, β-MSH, γ-MSH, and δ-MSH. Port Jackson shark δ-MSH has 83% primary sequence identity with dogfish and stingray δ-MSH, and it appears that the δ-MSH sequence may have been the result of an internal domain duplication and reinsertion of the β-MSH sequence. The presence of the δ-MSH sequence in the POMC genes of representatives of both superorders of infraclass Neoselachii would indicate that the δ-MSH sequence must have been present in the ancestral euselachian shark that gave rise to the neoselachian radiation.

Generation of a bioactive neuropeptide in a cell-free system

We have developed an in vitro assay for pre-pro-neuropeptide synthesis and processing. Mouse proopiomelanocortin (POMC) cDNA was cloned into a vector containing T7 promoter. In vitro transcription and translation were carried out to produce the proopiomelanocortin peptide. The pro-peptide was processed by incubating with cell extract of mouse and rat pituitary cell lines. The activity of processed mature peptide was tested in a cell line expressing human melanocortin 4 receptor (MC4R). Using this approach, we produced biologically active α-melanocyte-stimulating hormone (α-MSH). Furthermore, we developed a universal functional assay for G-protein-coupled receptors (GPCRs) using a reporter gene assay. More than 20 different GPCRs were examined using this functional assay. Our results demonstrated that the activity of all GPCRs could be measured by the functional assay. It should be possible to identify novel bioactive peptides for orphan GPCRs by the combination of in vitro processing and GPCR functional assays.

Molecular cloning of proopiomelanocortin (POMC) cDNA from mud turtle, Pelodiscus sinensis

The complete complementary DNA (cDNA) of proopiomelanocortin (POMC), a common precursor of opioid hormone β-endorphin, melanotropin (MSH), and corticortropin (ACTH), was cloned and sequenced from pituitary and hypothalamus of mud turtle ( Pelodiscus sinensis) by RT-PCR and rapid amplification of cDNA end (RACE) methods. Two transcripts of POMC mRNAs with different polyadenylation sites were observed. Both transcripts had an open reading frame encoding a 261-amino acid peptide containing nine dibasic amino acids (pair of Arg and Lys), putative proteolytic cleavage sites for processing to functional peptides. All the functional peptide fragments of mud turtle POMC, γ-MSH, α-MSH, ACTH, β-MSH, and β-endorphin, are flanked by dibasic residues as found in other tetrapods, implying that it could be processed to give rise to all members of POMC-derived peptides. The deduced amino acid sequences of mud turtle POMC displays 63–67% identity with amphibian, 59% with chicken, 48–53% with mammals, and 37–59% identity with fish. However, functional peptide fragments are much more conserved than overall sequence and intervening fragments. In addition to pituitary and brain, mud turtle POMC mRNAs are also expressed in many peripheral tissues, such as skin, thyroid, and testis. This is the first report on the complete sequence of cDNA nucleotides and deduced amino acids of POMC in reptile.

Cloning of a proopiomelanocortin cDNA from the pituitary gland of the sea bass (Dicentrarchus labrax) and assessment of mRNA expression in different tissues by means of real-time PCR.

Proopiomelanocortin (POMC) cDNA was cloned from sea bass (Dicentrarchus labrax) pituitary gland. A 743 nucleotide sequence was obtained coding for the following sequences flanked by sets of proteolytic cleavage sites: ACTH (Ser(88)-Met(127)), alpha-MSH (Ser(88)-Gly(102)), CLIP (Pro(106)-Met(127)), beta-LPH (Glu(131)-Gln(208)), gamma-LPH (Glu(131)-Ser(175)), beta-MSH (Asp(159)-Ser(175)), and beta-endorphin (Tyr(178)-Gln(208)). No region homologous to gamma-MSH/joining peptide (a tetrapod POMC feature) was found. Amino acid sequence identity was high with other teleostean species considered (tilapia: 73%) and lower with elasmobranchs (dogfish: 42%). However, the presumed biologically active peptides were highly conserved within all species considered: alpha-MSH (93-100%), ACTH (80-95%) and beta-endorphin (54-90%). Real-time PCR allowed us to quantify the expression of the POMC in different tIssues of the sea bass: pituitary gland, liver, gonad and head kidney. No significant POMC expression was found in the integument. In pituitary gland, gonads, head kidney and liver, POMC expression was respectively, 1.26x10(10), 2.67x10(5), 2.06x10(4) and 1.67x10(4) copies/ micro g mRNA.