Cloning, sequencing, and expression of H.a.YNR1 and H.a.YNI1, encoding nitrate and nitrite reductases in the yeast Hansenula anomala.
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 lambda EMBL3 genomic DNA library. As probe, a 3.2 kb DNA fragment isolated from a lambda gt11 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.
García-Lugo
P
P
Departamento de Bioquímica y Biología Molecular, Grupo del Metabolismo del Nitrógeno, Consejo Superior de Investigaciones Científicas, Universidad de La Laguna, E-38206 La Laguna, Tenerife, Canarias, Spain.
González
C
C
Perdomo
G
G
Brito
N
N
Avila
J
J
de La Rosa
J M
JM
Siverio
J M
JM
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000032:analysis; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics* / Q000378:metabolism; D009566:Nitrates / Q000378:metabolism; D009572:Nitrite Reductases / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000235:genetics* / Q000378:metabolism; D000644:Quaternary Ammonium Compounds / Q000378:metabolism; D012331:RNA, Fungal / Q000032:analysis; D017422:Sequence Analysis, DNA
D004271:DNA, Fungal; D009566:Nitrates; D000644:Quaternary Ammonium Compounds; D012331:RNA, Fungal; D009565:Nitrate Reductases; D009572:Nitrite Reductases; D050901:Nitrate Reductase