PCR cloning and expression of the molt-inhibiting hormone gene for the crab (Charybdis feriatus)

A PCR-based genomic DNA walking technique was used to clone the gene for th e molt-inhibiting hormone of the crab, Charybdis feriatus. Several overlapp ing genomic clones were isolated, and the MIH gene for the crab was reconst ructed. DNA sequence determination of the overlapping clone reveals that th e MIH gene spans 4.3 kb and consists of three exons and two introns. Exons 1 and 2 carry a coding sequence for the signal peptide, and exons 2 and 3 c onsist of coding sequence for the mature peptide. The exon-intron boundary of the crab MIH gene also follows the 'GT-AG rule' for the splice donor and acceptor. The deduced amino acid sequence of MIH shows the highest overall similarity to those of the crabs, Callinectes sapidus and Carcinus maenas, and the gonad-inhibiting hormone (GIH) of the lobster. The putative polyad enylation signal is approximately 1.0 kb 3' downstream of the termination c odon (TGA). Genomic Southern blot analysis indicates that few genomic fragm ents were hybridized to the cDNA probe. The 5' flanking region contains a p utative promoter with several putative cis elements similar to some vertebr ate neuropeptide genes. The 530-bp flanking region was subcloned separately to two promoterless reporter plasmids carrying either the Green Fluorescen t Protein gene (GFP) or the Choramphenicol Acetyltransferase gene (CAT). Th e DNA constructs were transfected into insect cells (Sf21) and mouse pituit ary cells (GH4ZR7), respectively. Green fluorescent protein was detected in some of the transfected insect cells, and expression of the CAT was detect ed in cells transfected with DNA constructs containing the crab promoter. B y RT-PCR, MIH transcripts can be detected in the eyestalk of shrimp in inte rmolt, early premolt, late premolt stages and females that brood their eggs . It can also be found in the brain, but not in the ovary, hepatopancreas, muscle and epidermis. During early larval development, MIH mRNA can be dete cted in the pre-hatched and the newly hatched larvae. Unlike the adult, the expression of the MIH in the larvae is exclusively in the brain. (C) 1998 Elsevier Science B.V. All rights reserved.