ISOLATION AND IN-SITU LOCALIZATION OF A CDNA-ENCODING A KEX2-LIKE PROHORMONE CONVERTASE IN THE NEMATODE CAENORHABDITIS-ELEGANS

1. A cDNA that encodes a Kex2-like prohormone convertase (PC) containi ng an active site similar to that of mammalian PC2 has been isolated f rom C. elegans. Total RNA was isolated from a mixed population of stra in BA713 worms. After poly-(A)-selection and reverse transcription, de generate/nested polymerase chain reactions (PCR) were performed using primers based on conserved regions within the active sites of the know n vertebrate and invertebrate endoproteases. 2. Two distinct 300-bp PC R products that shared homologies with the active sites of known Kex2- like endoproteases were isolated. These two PCR products were used to screen a C. elegans cDNA library. 3. The complete cDNA for a Kex2-like endoprotease, designated CELPC2, was isolated and determined to be 25 27 bp in length. This size was confirmed by northern analysis. The ded uced amino acid sequence for the CELPC2, cDNA is very similar to the k nown Kex2-like endoproteases, especially at conserved regions within t he active sites, but not identical to any one of them. The strongest s tructural homology was to vertebrate and invertebrate PC2 sequences. 4 . In situ hybridization suggests that CELPC2 is synthesized primarily in cells associated with the circumpharyngeal nerve ring and the dorso rectal ganglion.