Cloning, sequencing, and expression of H.a. YNR1 and H.a. YNI1, encoding nitrate and nitrite reductases in the yeast Hansenula anomala

Abstract

A single Hansenula anomala genomic DNA fragment containing the genes H.a. YNR1 (yeast nitrate reductase) and H.a. YNI1 (yeast nitrite reductase) encoding nitrate and nitrite reductase, respectively, was isolated from a λEMBL3 genomic DNA library. As probe, a 3.2 kb DNA fragment isolated from a λgt11 H. anomala genomic DNA library screened with antiserum anti-NR from H. anomala was used. H.a. YNR1 and H.a. YNI1 genes are separated by 473 bp and encode putative proteins of 870 and 1077 amino acids, respectively, with great similarity to nitrate and nitrite reductases from other organisms. Northern blot analysis revealed that both genes are highly expressed in nitrate, very low in nitrate plus ammonium, and no expression was detected in ammonium or nitrogen-free media. Levels of nitrate reductase and nitrite reductase were very low or undetectable by Western blot analysis in nitrogen-free and ammonium media, whereas both proteins were present in nitrate and ammonium plus nitrate media. The nucleotide sequence Accession No. is AF123281. Copyright (C) 2000 John Wiley and Sons, Ltd.