Cloning of metacaspase gene expressed in the opportunistic human pathogenic fungus, Aspergillus fumigatus

Introduction and objective: Caspases belong to a distinct class of cysteine proteases which also includes hemoglobinases, gingipains, clostripains, and separases, the proteases involved in chromosome segregation in mitosis and meiosis. Two families of predicted Caspase Homoglobinase Fold (CHF)-proteases were identified and shown to be more closely related to the caspases than to other proteases of this class and hence dubbed paracaspases and metacaspases. The aim of present study was to isolate genes particularly caspase-related ones up-regulated in the earliest stages of the stationary phase (up to four hours) and using clones to perform a PCR on cDNA prepared from exponential and stationary phase mRNA.

Materials and methods: After the use of the clorimetric assay to get evidence of caspase activity, both strands of cDNA were used for cloning the PCR products. The whole plasmid and freeze-dried material were sent for automated sequencing. By searching in NCBI homepage, particularly Blast part, all the alignment sequences were shown and then after similar genes were recognized as well.

Results: There was no sign of any cDNA in the midlog and 4h stationary phases which shows no RNA synthesis in those phases. The extended sequence of the gene was found to have a high level of identity (87%) with a metacaspase from Schizosaccharomyces pombe through a BLASTX search.

Conclusion: This information may be used to fabricate microarrays which would enable a genome-wide analysis of gene expression of Aspergillus fumigatus as it enters and during the stationary phase.