Cloning of the first cDNA encoding a putative CCRFamide precursor: identification of the brain, eyestalk ganglia, and ca
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Cloning of the first cDNA encoding a putative CCRFamide precursor: identification of the brain, eyestalk ganglia, and cardiac ganglion as sites of CCRFamide expression in the American lobster, Homarus americanus J. Joe Hull1 · Melissa A. Stefanek1 · Patsy S. Dickinson2 · Andrew E. Christie3 Received: 16 January 2020 / Accepted: 18 November 2020 / Published online: 26 November 2020 © This is a U.S. government work and not under copyright protection in the U.S.; foreign copyright protection may apply 2020
Abstract 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. Keywords Decapoda · Homarus americanus · In silico transcriptome mining · RT-PCR · Nervous system
Introduction Electronic supplementary material The online version of this article (https://doi.org/10.1007/s10158-020-00257-z) contains supplementary material, which is available to authorized users. * J. Joe Hull [email protected] 1
Pest Management and Biocontrol Research Unit, US Arid Land Agricultural Research Center, USDA Agricultural Research Services, 21881 North Cardon Lane, Maricopa, AZ 85138, USA
2
Department of Biology, Bowdoin College, 6500 College Station, Brunswick, ME 04011, USA
3
Békésy Laboratory of Neurobiology, Pacific Biosciences Research Center, School of Ocean and Earth Science and Technology, University of Hawaii at Manoa,
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