Clustering of the YNA1 gene encoding a Zn(II)2Cys6 transcriptional factor in the yeast Hansenula polymorpha with the nitrate assimilation genes YNT1, YNI1 and YNR1, and its involvement in their transcriptional activation

The genes encoding the nitrate transporter (YNT1), nitrite reductase (YNI1) and nitrate reductase (YNR1) are clustered in the yeast Hansenula polymorpha. In addition, DNA sequencing of the region containing these genes demonstrated that a new open reading frame called YNA1 (yeast nitrate assimilation) was located between YNR1 and YNI1. The YNA1 gene encodes a protein of 529 residues belonging to the family of Zn(II)2Cys6 fungal transcriptional factors, and has the highest similarity to the transcriptional factors encoded by nirA, and to a smaller extent to nit-4, involved in the nitrate induction of the gene involved in the assimilation of this compound in filamentous fungi. Northern blot analysis showed the presence of the YNA1 transcript in cells incubated in nitrate, nitrate plus ammonium, ammonium, and nitrogen-free media, with a decrease in its levels in those cells incubated in ammonium. In nitrate the strain Δyna1::URA3, with a disrupted YNA1 gene, neither grew nor expressed the genes YNT1, YNI1 and YNR1. In the gene cluster YNT1-YNI1-YNA1-YNR1, the four genes were transcribed independently in the YNT1 → YNR1 direction and the transcription start sites were determined by primer extension.