Characterization and nitrogen-source regulation at the transcriptional level of the gdhA gene of Aspergillus awamori encoding an NADP-dependent glutamate dehydrogenase.
A 28.7-kb DNA region containing the gdhA gene of Aspergillus awamori was cloned from a genomic DNA library. A fragment of 2570 nucleotides was sequenced that contained ORF1, of 1380 bp, encoding a protein of 460 amino acids (Mr 49.4 kDa). The encoded protein showed high similarity to the NADP-dependent glutamate dehydrogenases of different organisms. The cloned gene was functional since it complemented two different Aspergillus nidulans gdhA mutants, restoring high levels of NADP-dependent glutamate dehydrogenase to the transformants. The A. awamori gdhA gene was located by pulsed-field gel electrophoresis in a 5.5-Mb band (corresponding to a doublet of chromosomes II and III), and was transcribed as a monocistronic transcript of 1.7 kb. Transcript levels of the gdhA gene were very high during the rapid growth phase and decreased drastically after 48 h of cultivation. Very high expression levels of the gdhA gene were observed in media with ammonium or asparagine as the nitrogen source, whereas glutamic acid repressed transcription of the gdhA gene. These results indicate that expression of the gdhA gene is subject to a strong nitrogen regulation at the transcriptional level.
Cardoza
R E
RE
Instituto de Biotecnología INBIOTEC, Parque Científico de León, Avda. del Real, 1, E-24006 León, Spain.
Moralejo
F J
FJ
Gutiérrez
S
S
Casqueiro
J
J
Fierro
F
F
Martín
J F
JF
D000595:Amino Acid Sequence; D001230:Aspergillus / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D015966:Gene Expression Regulation, Fungal / Q000187:drug effects; D015723:Gene Library; D005969:Glutamate Dehydrogenase / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D009249:NADP / Q000494:pharmacology*; D009584:Nitrogen / Q000378:metabolism* / Q000494:pharmacology; D016366:Open Reading Frames; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000032:analysis; D015183:Restriction Mapping; D016415:Sequence Alignment; D014158:Transcription, Genetic; D014170:Transformation, Genetic
D004271:DNA, Fungal; D012333:RNA, Messenger; D009249:NADP; D005969:Glutamate Dehydrogenase; D009584:Nitrogen