Detection of a protein, AngCP, which binds specifically to the three upstream regions of glaA gene in A. niger T21

Electromobility shift assay (EMSA) was used to scan 600 bp of 5′ cis regulatory sequence of Aspergillus niger (A. niger) T21 glucoamylase gene (glaA) for binding by partially fractionated T21 protein extracted from starch-induced mycelia. In this process, one protein, AngCP, was detected to bind specifically to three regions covering -374 to -344, -484 to -414 and -580 to -540 relative to the glaA translational start codon. UV-crosslinking of DNA-protein complex showed that MW of AngCP was 10 ku. DNase I footprinting analysis demonstrated that AngCP specifically binds to two CCAAT containing sequences within the regions between -374 and -344 and -484 and -414 bp. And the region between -580 and -540 bp contains CCAAT similar box, CCTAT. The results indicated that AngCP is probably one of the members of CCAAT-binding protein families, which are generally involved in enhancement of gene expression in filamentous fungi. These findings suggested that AngCP should be a transcription activator for high-level expression of glaA gene.