ABSTRACT
A novel cellulase [beta-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA belonging to glycoside hydrolase family (GHF) 45 was cloned from the mulberry longicorn beetle, Apriona germari. The cDNA encoding EGase of A. germari (Ag-EGase) is 711 bp long with an open reading frame of 237 amino acid residues. The Ag-EGase was closely related to another beetle, Phaedon cochleariae, cellulase and one symbiotic protist cellulase in the hindgut of the termite Reticulitermes speratus, those belonging to GHF 45. The catalytic sites of GHF 45 are conserved in Ag-EGase. Southern blot analysis of genomic DNA suggested the presence of Ag-EGase gene as a single copy and Northern blot analysis confirmed midgut-specific expression at transcriptional level. Similarly, the Ag-EGase enzyme assay exhibited high activity only in midgut tissue, suggesting that the midgut is the prime site where large quantities of EGase are synthesized for degrading the absorbed cellulose from the diet. The cDNA encoding Ag-EGase was expressed as a 29-kDa polypeptide in baculovirus-infected insect Sf9 cells and the culture supernatants of the recombinant baculovirus-infected cells showed EGase enzyme activity of 15.25 U/ml of medium containing 0.5 x 10(6) cells at 5 days post-infection (p.i.). The enzyme activity of the purified recombinant Ag-EGase expressed in baculovirus-infected insect cells was approximately 992 U per mg of recombinant Ag-EGase. The purified recombinant Ag-EGase showed the highest enzymatic activity at 50 degrees C and pH 6.0, and was stable at 55 degrees C at least for 10 min.
Subject(s)
Cellulase/genetics , Cellulase/metabolism , Coleoptera/enzymology , Coleoptera/genetics , DNA, Complementary/genetics , Amino Acid Sequence , Animals , Cellulase/chemistry , Cellulase/isolation & purification , Cloning, Molecular , Gene Expression Profiling , Genes, Insect/genetics , Genomics , Larva/enzymology , Larva/genetics , Molecular Sequence Data , Organ Specificity , Phylogeny , Recombinant Proteins/genetics , Recombinant Proteins/isolation & purification , Recombinant Proteins/metabolism , Sequence Alignment , Sequence Analysis, DNA , Sequence Homology, Amino AcidABSTRACT
A cDNA encoding a defender against apoptotic cell death (DAD1) homologue was cloned from a cDNA library of the spider, A. ventricosus. Sequence analysis of the cDNA encoding the DAD1 homologue of A. ventricosus revealed that the 339-bp cDNA has an open reading frame of 113 amino acid residues. The deduced amino acid sequence of the A. ventricosus DAD1 homologue showed 75.4% of identity to Drosophila melanogaster, 74.6% of identity to Xenopus laevis, and 73.1% of identity to Homo sapiens, Sus scrofa, Mesocricetus auratus, Rattus norvegicus and Mus musculus. All animal DAD1 including A. ventricosus DAD1 homologue formed a subgroup, excluding all plant DAD1 proteins in the phylogenetic analysis. Northern blot analysis shows that the transcripts of A. ventricosus DAD1 homologue gene are present in all tissues examined, suggesting that A. ventricosus DAD1 is expressed in most, if not all, body tissues. Interestingly, the transcript levels of A. ventricosus DAD1 homologue gene were particularly high when exposed at low (4 degrees C) and high (37 degrees C) temperatures, suggesting that the gene is responsive to temperature stimuli.
