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) containing an active site similar to that of mammalian PC2 has been isolated from C. elegans. Total RNA was isolated from a mixed population of strain BA713 worms. After poly-(A)-selection and reverse transcription, degenerate/nested polymerase chain reactions (PCR) were performed using primers based on conserved regions within the active sites of the known vertebrate and invertebrate endoproteases. 2. Two distinct 300-bp PCR 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 2527 bp in length. This size was confirmed by northern analysis. The deduced amino acid sequence for the CELPC2 cDNA is very similar to the known Kex2-like endoproteases, especially at conserved regions within the active sites, but not identical to any one of them. The strongest structural 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 dorsorectal ganglion.
Gómez-Saladín
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Department of Physiology and Biophysics, University of Miami School of Medicine, Florida 33101.
Wilson
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Dickerson
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D017173:Caenorhabditis elegans / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D017931:DNA Primers; D018076:DNA, Complementary / Q000032:analysis / Q000302:isolation & purification; D018509:DNA, Helminth / Q000378:metabolism; D017952:Ganglia, Invertebrate / Q000378:metabolism*; D015723:Gene Library; D006801:Humans; D017403:In Situ Hybridization; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D043484:Proprotein Convertases; D018510:RNA, Helminth / Q000302:isolation & purification; D029701:Saccharomyces cerevisiae Proteins; D017386:Sequence Homology, Amino Acid; D013381:Subtilisins / Q000096:biosynthesis* / Q000235:genetics
D017931:DNA Primers; D018076:DNA, Complementary; D018509:DNA, Helminth; D018510:RNA, Helminth; D029701:Saccharomyces cerevisiae Proteins; D043484:Proprotein Convertases; D013381:Subtilisins; C058751:KEX2 protein, S cerevisiae