Cloning and characterization of the 5'-flanking region of the oxalate decarboxylase gene from Flammulina velutipes

Abstract

The oxalate-degrading enzyme, oxalate decarboxylase (OXDC), was purified and characterized from Flammulina velutipes, a basidiomycetous fungus [Mehta and Datta (1991) J. Biol. Chem. 266, 23548—23553]. The cDNA cloning and analyses revealed that OXDC transcription was induced by oxalic acid. However, in this report, we show that OXDC transcription is induced by low pH, not by oxalate. To understand the regulatory mechanism of OXDC expression, we have cloned and analysed a 580-bp genomic fragment from the 5′-flanking region of the OXDC gene. Sequence analysis showed the presence of several eukaryotic transcription factor binding motifs within the −580bp of the upstream region. Electrophoretic-mobility-shift assays with partially purified cell extracts revealed specific binding of a factor in acid-induced, but not in uninduced, extracts. Furthermore, DNase I protection assays using the partially purified fraction from oxalic acid-induced extract revealed a footprint of a 13-bp sequence 5′GCGGGGTCGCCGA3′, termed low pH responsive element (LPRE), corresponding to the −287 to −275bp region of the OXDC promoter. Our results suggest that in F. velutipes cells, activation of OXDC transcription in response to low pH is mediated by the binding of a novel transcription factor through the LPRE site in the OXDC promoter.