Lee J, Lecaude S, Danielson P, Sollars C, Alrubaian J, Propper C R, Lihrmann I, Vaudry H, Dores R M
Department of Biological Sciences, University of Denver, Colo., USA.
Neuroendocrinology. 1999 Jul;70(1):43-54. doi: 10.1159/000054458.
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.
使用针对阿片样物质核心序列YGGFM的简并引物，从非洲肺鱼（Protopterus annectens）的大脑以及西部锄足蟾（Spea multiplicatus）的大脑中克隆并测序阿黑皮素原（POMC）cDNA。此外，使用阿片样物质特异性引物克隆并测序了与西部锄足蟾前脑啡肽开放阅读框一部分相对应的3'RACE产物。对于这两个物种，均从单个大脑中制备cDNA，并且阿片样物质特异性简并引物为检测阿片样物质相关cDNA提供了可靠的探针。非洲肺鱼POMC cDNA长度为1168个核苷酸，包含与四足动物POMC和鱼类POMC相似的区域。非洲肺鱼POMC编码一个类似四足动物的γ-MSH序列，其两侧是成对的碱性氨基酸蛋白水解切割位点。硬骨鱼POMC中的γ-MSH区域要么具有简并切割位点，要么在某些物种中完全不存在。然而，非洲肺鱼的γ-MSH序列确实包含一个在四足动物γ-MSH序列中未观察到的缺失。肺鱼POMC的β-内啡肽区域在N端区域具有色氨酸-天冬氨酸二氨基酸序列，并且在β-内啡肽的C端区域有一个额外的谷氨酸残基——这些特征在鱼类β-内啡肽中存在，但在四足动物β-内啡肽中不存在。西部锄足蟾POMC长度为1186个核苷酸，呈现出四足动物POMC典型的组织结构。然而，蟾蜍POMC在β-MSH序列N端确实缺少一个成对的碱性氨基酸蛋白水解切割位点。因此，与大鼠POMC一样，β-MSH是否是蟾蜍大脑或垂体中间叶的终产物值得怀疑。在氨基酸水平上，蟾蜍POMC与非洲爪蟾POMC的序列同一性为76%，与泽陆蛙POMC的序列同一性为68%。将讨论如何利用这些POMC序列评估无尾两栖动物内部的系统发育关系。关于西部锄足蟾前脑啡肽cDNA片段，在该片段中发现编码了两个甲硫脑啡肽序列和甲硫脑啡肽-RF序列。正如在非洲爪蟾和泽陆蛙前脑啡肽中所见，在西部锄足蟾前脑啡肽的C端区域未检测到亮脑啡肽序列。亮脑啡肽序列的缺失可能是无尾两栖动物前脑啡肽的一个共同特征。
