Over the past decade, many new peptide families have been identified via in silico analyses of genomic and transcriptomic datasets. While various molecular and biochemical methods have confirmed the existence of some of these new groups, others remain in silico discoveries of computationally assembled sequences only. An example of the latter are the CCRFamides, named for the predicted presence of two pairs of disulfide bonded cysteine residues and an amidated arginine-phenylalanine carboxyl-terminus in family members, which have been identified from annelid, molluscan, and arthropod genomes/transcriptomes, but for which no precursor protein-encoding cDNAs have been cloned. Using routine transcriptome mining methods, we identified four Homarus americanus (American lobster) CCRFamide transcripts that share high sequence identity across the predicted open reading frames but more limited conservation in their 5′ terminal ends, suggesting the Homarus gene undergoes alternative splicing. RT-PCR profiling using primers designed to amplify an internal fragment common to all of the transcripts revealed expression in the supraoesophageal ganglion (brain), eyestalk ganglia, and cardiac ganglion. Variant specific profiling revealed a similar profile for variant 1, eyestalk ganglia specific expression of variant 2, and an absence of variant 3 expression in the cDNAs examined. The broad distribution of CCRFamide transcript expression in the H. americanus nervous system suggests a potential role as a locally released and/or circulating neuropeptide. This is the first report of the cloning of a CCRFamide-encoding cDNA from any species, and as such, provides the first non-in silico support for the existence of this invertebrate peptide family.

在过去的十年中，通过对基因组和转录组数据集的计算机分析鉴定了许多新的肽家族。虽然各种分子和生化方法已经证实了其中一些新组的存在，但其他组仍停留在计算机组装序列的计算机发现中。后者的一个例子是 CCRFamides，因预测在家族成员中存在两对二硫键半胱氨酸残基和酰胺化精氨酸-苯丙氨酸羧基末端而命名，这些已从环节动物、软体动物和节肢动物基因组/转录组中鉴定出来，但尚未克隆出编码前体蛋白的 cDNA。使用常规转录组挖掘方法，我们鉴定了四种美洲白鹤（美洲龙虾）CCRFamide 转录本，它们在预测的开放阅读框中具有较高的序列同一性，但其 5' 末端的保守性更为有限，表明白鹤基因经历了选择性剪接。使用设计用于放大所有转录物共有的内部片段的引物进行 RT-PCR 分析，揭示了食管上神经节（大脑）、眼柄神经节和心脏神经节中的表达。变体特异性分析揭示了变体 1 的相似特征、变体 2 的眼柄神经节特异性表达以及所检查的 cDNA 中不存在变体 3 的表达。 CCRFamide 转录本表达在美洲人神经系统中的广泛分布表明其作为局部释放和/或循环神经肽的潜在作用。这是从任何物种克隆 CCRFamide 编码 cDNA 的第一份报告，因此为该无脊椎动物肽家族的存在提供了第一个非计算机支持。