The chemorepellent semaphorin is expressed in the horseshoe crab, Limulus polyphemus

Abstract

Semaphorins are a family of soluble and membrane-bound proteins that play a critical role in axonal guidance and other processes of neuronal development. Currently, more than twenty semaphorins have been identified, all of which share a conserved 500 amino acid domain near the amino terminus. Semaphorins are divided into eight classes according to species of origin and structural similarities. Classes 1 and 2 are found in invertebrates, classes 3 through 7 are present in vertebrates and viruses encode class V semaphorin. Microarray analysis of Limulus CNS RNA revealed the presence of a semaphorin-like gene in Limulus polyphemus. Based on these data, we aligned 31 different sequences and designed degenerate primers for the consensus domains (WTT/SFLKA) and (DPY/VCA/GW). RT-PCR products were generated using 6 forward primers and 4 reverse primers. The expected size PCR products (750 bp) was obtained and then ligated with pCR II TOPO vector and transferred into E. coli Top 10. Five partial semaphorin cDNAs were found in Limulus: semaphorins 1a, 1b, 2a, 2b and F (now known as 5) were partially cloned. Subsequent Northern blot analyses using these Limulus specific-probes revealed hybridization with total RNAs purified from six different tissues.