Fig. 5. Expression of NO synthase (Gl-NOS) in land crab tissues. Total RNA was DNase-treated, reverse-transcribed and PCR-amplified using sequence-specific primers (see Materials and methods). Shown are inverse images of ethidium bromide-stained agarose gels of the PCR products. (A) First-round PCR generated a product of the expected size (2.1 kb). Gl-NOS was expressed in testis (Tt), gill (Gi), ovary (Ov), eyestalk neural ganglia (EG) and Y-organ (YO). Gl-NOS mRNA was not detected in integument (Ig), thoracic ganglion (TG), digestive gland (DG), heart (Ht) or claw muscle (CM). (B) Nested PCR of the initial PCR generated a product of the expected size (800 bp), which confirmed the identity of the initial product as Gl-NOS. In other experiments, a Gl-NOS product was obtained from thoracic ganglion (data not shown). (C) Elongation factor 2 (EF2) served as internal positive control, as it was constitutively expressed in all tissues. Reactions without template [water (W) lane k] served as a negative control. Positions of DNA size markers are indicated on the left.