Worm Breeder's Gazette 11(3): 20
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Cysteine (thiol) proteases are found throughout nature and are involved in a wide variety of biological processes. C. elegans extracts have been shown by Sarkis et al. to contain two distinct cysteine protease activities (1). Although these enzymes comigrate with lysosomal markers in isopycnic centrifugation (2), their functions are unknown. In addition, Sakanari et al. have recently reported using degenerate oligonucleotide primers to amplify a 160 bp cysteine protease gene fragment from C. elegans in the Polymerase Chain Reaction (PCR) (3). As a first step in characterizing the functions and regulation of expression of the C. elegans cysteine proteases, we have used the 160 bp PCR fragment to probe a mixed-stage C. elegans cDNA library (from Cynthia Kenyon's laboratory). Five clones were identified, subcloned, and partially sequenced. All of the clones contained the same nucleotide sequence and there was no evidence of trans-splicing. The predicted amino acid sequence was 53% homologous to that of rat cathepsin B and contained a highly conserved region flanking the active site cysteine residue. Like those of other eukaryotes, the C. elegans cysteine protease appears to be made as a proenzyme and has 84 amino acids in its pro region. In agreement with the biochemical data of Sarkis et al., we have molecular biological evidence that C. elegans contains at least two cysteine proteases. First, the nucleotide sequence of the cDNA clones is only 71% homologous to that of the 160 bp PCR fragment, and the two predicted amino acid sequences are only 64% homologous. This difference is too great to be accounted for by misincorporation errors during the PCR reaction. Second, preliminary Southern analysis shows that the PCR fragment and a fragment of the cDNA clone recognize bands of different size in restriction digested C. elegans genomic DNA. That the PCR fragment did not recognize a cDNA clone containing its parent gene could be due to the gene being transcribed at low levels or only at specific stages in the C. elegans life cycle. This would lead to underrepresentation of the gene in a mixed-stage cDNA library. We are currently examining the transcription patterns of the two protease genes by Northern analysis using the PCR fragment and part of the cDNA clone as hybridization probes. In addition, we are using these same probes to isolate genomic clones for the two genes from a lambdaEMBL3 library obtained from Cynthia Kenyon. These experiments will lead to a more thorough characterization of the functions and regulation of the C. elegans cysteine proteases.