Transcriptional analysis of two endoinulinase genes inuA and inuB in Aspergillus niger and nucleotide sequences of their promoter regions

Aspergillus niger 12 contained two copies of endoinulinase genes (inuA and inuB) in the genome (K. Ohta ct al., Biosci. Biotechnol. Biochem., 62, 1731-1738, 1998). The inuA- and inuB-specific DNA probes were constructed according to the respective 3'-noncoding sequences that diverged from each other. Poly(A)(+) RNA was prepared from mycelia grown on inulin, fructose, or glucose in submerged culture. Three endoinulinase cDNA sequences that corresponded to the coding regions and their 5'- and S'-flanking regions were obtained by reverse transcription and subsequent polymerase chain reaction. Southern blot analysis revealed that the amplified cDNA 3'-noncoding sequences hybridized to the inuB probe but not to the inuA probe, regardless of the carbon source. The data suggest that only the inuB gene was transcribed constitutively. Four distinct 5' ends of the transcripts were observed at positions -80 (A), -72 (G), -69 (A), and -65 (A) from the start codon. The inuB mRNAs were polyadenylated at various sites between 94 and 297 bp downstream of the stop codon. We have determined the nucleotide sequences of the 1201- and 1017-bp 5'-noncoding regions of the inuA and inuB genes, respectively. The inuB promoter region included a putative TATA box at - 116 (TATATA).