cDNA cloning of a salivary chymotrypsin-like protease and the identification of six additional cDNAs encoding putative digestive proteases from the green mirid, Creontiades dilutus (Hemiptera: Miridae).
RT-PCR with degenerate primers was used to amplify partial cDNA fragments for one serine protease gene and three cysteine protease genes from poly(A) RNA isolated from the midgut of the green mirid, Creontiades dilutus. The serine protease amplicon showed homology to insect trypsin-like protease genes, and all three cysteine protease amplicons showed homology to cathepsin L-like protease genes.RT-PCR was also used to amplify fragments of three serine protease genes from salivary gland poly(A) RNA. One of these salivary gland serine protease amplicons was used to screen a whole organism cDNA library to isolate a full length cDNA clone, designated CdSp1 (Accession AY055753), which encodes a putative chymotrypsin-like protease. CdSp1 codes for a 293 amino acid protein that contains a signal peptide and activation peptide, as well as the catalytic triad present in all serine proteases and several of the binding pocket residues characteristic of chymotrypsins. In situ hybridisation showed that the transcript is expressed in the posterior lobe of the principal salivary gland, but not in the anterior lobe of the principal salivary gland, the accessory salivary gland or the midgut.
Colebatch
G
G
Division of Entomology, CSIRO, Canberra, ACT, Australia. gillian.colebatch@csiro.au
Cooper
P
P
East
P
P
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D053818:Chymases; D003001:Cloning, Molecular; D003546:Cysteine Endopeptidases / Q000235:genetics; D018076:DNA, Complementary; D010450:Endopeptidases / Q000235:genetics; D015870:Gene Expression; D006430:Hemiptera / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction / Q000379:methods; D012469:Salivary Glands / Q000201:enzymology; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000235:genetics*
D018076:DNA, Complementary; D010450:Endopeptidases; D012697:Serine Endopeptidases; D053818:Chymases; D003546:Cysteine Endopeptidases