BATISTA, L.A.; KIM, H.W.; LEE, K.J.; MYKLES, D.L.; Colorado State University, Fort Collins: Cloning and tissue expression of nitric oxide synthase in land crab (Gecarcinus lateralis)

Nitric oxide (NO) has been implicated in a variety of physiological processes, including the modulation of neural networks in vertebrates and invertebrates. The neuronal isoform of nitric oxide synthase (NOS) is a calcium/calmodulin-activated enzyme that generates NO through the conversion of arginine to citrulline. NO activates a NO-sensitive guanylyl cyclase, resulting in an increase in intracellular cGMP. In efforts to further study the tissue distribution of NOS in crustaceans, a cDNA encoding a full-length sequence of NOS (open reading frame ~3.6 kb) was obtained by RT-PCR and 3′ and 5′ RACE from Y-organ (YO) and thoracic ganglion mRNA. The deduced Gl-NOS sequence (1,119 amino acids; estimated mass 129.6 kDa) was 55% identical to the NOS sequence from the mosquito Anopheles gambiae. RT-PCR was used to analyze the expression of NOS in intermolt land crab tissues. Highest levels of NOS mRNA were found in testis, gill, ovary, and eyestalk (ES) ganglia. Other results using RT-PCR with nested primer pairs showed that NOS was expressed at lower levels in heart, YO, and thoracic ganglion. Studies are now in progress to quantify the effects of elevated molting hormones (ecdysteroids) on NOS expression using real-time RT-PCR. Supported by NSF (IBN-9904528).