Mahon A C, Lloyd P E, Weiss K R, Kupfermann I, Scheller R H
Proc Natl Acad Sci U S A. 1985 Jun;82(11):3925-9. doi: 10.1073/pnas.82.11.3925.
We have identified cells in the central nervous system of the marine mollusc Aplysia that react with antibody raised against the small cardioactive peptide B (SCPB). Antisera to this neuropeptide stained a subset of central neurons that include the large identified buccal neurons, B1 and B2. The distribution of SCP-containing neurons was used in a strategy to isolate a cDNA clone encoding the precursor protein for the peptide. RNA from neurons B1 and B2 and from cells that did not stain with SCPB antisera was used to direct the synthesis of radiolabeled cDNA probes. A cDNA clone complimentary to mRNA specifically expressed in the B1 and B2 cells was isolated by differentially screening a buccal cDNA library with these probes. The cloned cDNA segment is 1394 nucleotides in length and contains a 408-base-pair open reading frame. The predicted precursor protein is composed of 136 amino acids and has a characteristic hydrophobic leader sequence. The sizes of the precursor protein with and without this leader sequence agree with in vivo and in vitro labeling studies. The amino acid sequences for SCPB and a related peptide, SCPA, are present and are flanked by known proteolytic processing sites.
我们已在海洋软体动物海兔的中枢神经系统中鉴定出一些细胞,它们能与针对小的心脏活性肽B(SCPB)产生的抗体发生反应。针对这种神经肽的抗血清对一部分中枢神经元进行了染色,这些神经元包括已明确的大型颊神经元B1和B2。含SCP的神经元分布情况被用于一种策略,以分离编码该肽前体蛋白的cDNA克隆。来自神经元B1和B2以及未被SCPB抗血清染色的细胞的RNA,被用于指导放射性标记cDNA探针的合成。通过用这些探针差异筛选颊cDNA文库,分离出了一个与在B1和B2细胞中特异性表达的mRNA互补的cDNA克隆。克隆的cDNA片段长度为1394个核苷酸,包含一个408个碱基对的开放阅读框。预测的前体蛋白由136个氨基酸组成,并有一个特征性的疏水前导序列。带有和不带有该前导序列的前体蛋白大小与体内和体外标记研究结果相符。SCPB和一种相关肽SCPA的氨基酸序列存在,且两侧为已知的蛋白水解加工位点。