Amplification and characterization of cysteine proteinase genes from nematodes.
In order to isolate proteinase genes from parasitic nematodes by polymerase chain reaction (PCR) techniques, we employed a pair of consensus oligonucleotide primers designed to anneal to the active site cysteine (primer ncpC) and asparagine (primer ncpN) coding regions of cysteine proteinases. The primers were biased toward the nucleotide and codon usages of cysteine proteinase genes of nematodes and were based on the consensus nucleotide sequences flanking the active site residues of genes from Haemonchus contortus, Caenorhabditis elegans, and Ostertagia ostertagi. We employed 'touchdown' PCR conditions and were able to amplify novel cysteine proteinase gene fragments from the rodent parasite Strongyloides ratti, the human pathogen S. stercoralis, the canine hookworm Ancylostoma caninum, and from C. elegans. These clones are gene homologs of cathepsin B-like (lysosomal associated) proteases and will facilitate screening of both cDNA and genomic DNA libraries.
Harrop
S A
SA
Molecular Parasitology Unit, Queensland Institute of Medical Research, Herston, Australia.
Prociv
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Brindley
P J
PJ
D000595:Amino Acid Sequence; D000722:Ancylostoma / Q000201:enzymology / Q000235:genetics; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics; D016384:Consensus Sequence; D003546:Cysteine Endopeptidases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D018076:DNA, Complementary; D004285:Dogs; D015723:Gene Library; D017238:Genes, Helminth; D006189:Haemonchus / Q000201:enzymology / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009348:Nematoda / Q000201:enzymology / Q000235:genetics*; D010028:Ostertagia / Q000201:enzymology / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D012377:Rodentia; D017386:Sequence Homology, Amino Acid; D013321:Strongyloides / Q000201:enzymology / Q000235:genetics
D018076:DNA, Complementary; D003546:Cysteine Endopeptidases