Cloning of proopiomelanocortin from the brain of the African lungfish, Protopterus annectens, and the brain of the western spadefoot toad, Spea multiplicatus
Cloning of proopiomelanocortin from the brain of the African lungfish, Protopterus annectens, and the brain of the western spadefoot toad, Spea multiplicatus
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DOI：
10.1159/000054458
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发表时间：
1999-07-01
影响因子：
4.1
通讯作者：
Dores, RM
中科院分区：
文献类型：
--
作者：
Lee, J;Lecaude, S;Dores, RM
利用阿片核心序列YGGFM特异的退化引物，克隆了非洲肺鱼（Protopterus annectens）和西方掌足蟾蜍（Spea multiplicatus）脑中的POMC (proopiomelanocortin) cdna，并对其进行了测序。此外，利用阿片特异性引物克隆了一个3'RACE产物，并对其进行了测序，该产物与S. multiplicatus proenkephalin开放阅读框的一部分相对应。对于这两个物种，cDNA都是由一个大脑合成的，一个退化的阿片特异性引物为检测阿片相关cDNA提供了可靠的探针。非洲肺鱼POMC cDNA全长1168个核苷酸，包含与四足动物POMC和鱼类POMC相似的区域。非洲肺鱼的POMC编码一个类似四足动物的γ - msh序列，该序列的两侧是成对的碱性氨基酸蛋白水解裂解位点。射线鳍鱼POMCs中的γ - msh区域要么具有退化的卵裂位点，要么在某些物种中完全不存在。然而，非洲肺鱼γ - msh序列确实包含一个缺失，这在四足动物γ - msh序列中没有观察到。-内啡肽区域在肺鱼POMC的n端具有二氨基酸序列色氨酸-天冬氨酸，在-内啡肽的c端具有额外的谷氨酸残基，这些特征在鱼的-内啡肽中发现，但在四足动物的-内啡肽中没有。西部掌足蟾蜍的POMC长度为1186个核苷酸，具有典型的四足动物POMC组织结构。然而，蟾蜍POMC确实缺少与β - msh序列配对的碱性氨基酸蛋白水解裂解位点n端。因此，像大鼠POMC一样，β - msh是否是蟾蜍脑或中间垂体的最终产物是值得怀疑的。在氨基酸水平上，蟾蜍POMC与非洲爪蟾（Xenopus laevis）的序列同源性为76%，与河狸蛙（Rana ribidunda）的序列同源性为68%。使用这些POMC序列来评估无脊椎两栖动物的系统发育关系将被讨论。在S. multiplicatus proenkephalin cDNA片段中，发现了两个metenkephalin序列和metenkephalin- rf序列。与X. laevis和R. ridibunda proenkephalin一样，在S. multiplicatus proenkephalin的c端区未检测到proenkephalin序列。脑啡肽序列的缺失可能是两栖动物脑啡肽的共同特征。
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, proenkephalin 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.