CLONING AND EXPRESSION OF 2 CRUSTACEAN HYPERGLYCEMIC-HORMONE MESSENGER-RNAS IN THE EYESTALK OF THE CRAYFISH ORCONECTES-LIMOSUS

Crustacean hyperglycemic hormone (CHH) is a multifunctional neurohormo ne produced in the eyestalk of crustaceans and is primarily involved i n the regulation of carbohydrate metabolism. In several crustacean spe cies, CHH isoforms with identical amino acid sequences and molecular m asses, but with different chromatographic elution patterns, are synthe sized. To obtain sequence information on the CHH preprohormone in the crayfish Orconectes limosus we isolated two full-length cDNAs encoding two structurally different preproCHH species. The sequences of these precursors differ slightly in the signal peptide, the CHH-precursor-re lated peptide(CPRP)-coding sequences and in the non-coding regions, bu t are identical in the CHH peptide-coding sequence. Determination of t he levels of preproCHH mRNAs and the amount of CHH peptide in the eyes talks of individual animals revealed that the ratio between the two pr eproCHH mRNAs varies for different individuals while the ratio between the two CHH peptide isoforms does not differ among animals. Our resul ts suggest that the existence of two CHH isoforms in the crayfish O. l imosus is due to a post-translational modification event. Northern-blo t analysis showed only one band in eyestalk tissue with a size of appr oximately 2.4 kb, similar to the sizes of the cDNA sequences. Southern -blot analysis revealed the presence of at least two preproCHH genes i n the crayfish suggesting a gene duplication event. Slight modificatio ns in the duplicated genes could be responsible for the existence of t he two preproCHH-encoding mRNAs.