Biomedical subjects

I Nikolaev

Publications and source records attributed to I Nikolaev.

6 recordsLinked to original sources

PubMed2002

Heterologous expression of the Aspergillus nidulans alcR-alcA system in Aspergillus niger.

The inducible and strongly expressed alcA gene encoding alcohol dehydrogenase I from Aspergillus nidulans was transferred together with the activator gene alcR, in the industrial fungus Aspergillus niger. This latter organism does not possess an inducible alc system but has an endogenously constitutive lowly expressed alcohol dehydrogenase activity. The overall induced expression of the alcA gene was of the same order in both fungi, as monitored by alcA transcription, alcohol dehydrogenase activity and heterologous expression of the reporter enzyme, beta-glucuronidase. However, important differences in the pattern of alcA regulation were observed between the two fungi. A high basal level of alcA transcription was observed in A. niger resulting in a lower ratio of alcA inducibility. This may be due to higher levels of the physiological inducer of the alc regulon, acetaldehyde, from general metabolism in A. niger which differs from that of A. nidulans.

Alcohol Dehydrogenase↗

PubMed2001

Ethanol catabolism in Aspergillus nidulans: a model system for studying gene regulation.

This article reviews our knowledge of the ethanol utilization pathway (alc system) in the hyphal fungus Aspergillus nidulans. We discuss the progress made over the past decade in elucidating the two regulatory circuits controlling ethanol catabolism at the level of transcription, specific induction, and carbon catabolite repression, and show how their interplay modulates the utilization of nutrient carbon sources. The mechanisms featuring in this regulation are presented and their modes of action are discussed: First, AlcR, the transcriptional activator, which demonstrates quite remarkable structural features and an original mode of action; second, the physiological inducer acetaldehyde, whose intracellular accumulation induces the alc genes and thereby a catabolic flux while avoiding intoxification; third, CreA, the transcriptional repressor mediating carbon catabolite repression in A. nidulans, which acts in different ways on the various alc genes; Fourth, the promoters of the structural genes for alcohol dehydrogenase (alcA) and aldehyde dehydrogenase (aldA) and the regulatory alcR gene, which exhibit exceptional strength compared to other genes of the respective classes. alc gene expression depends on the number and localization of regulatory cis-acting elements and on the particular interaction between the two regulator proteins, AlcR and CreA, binding to them. All these characteristics make the ethanol regulon a suitable system for induced expression of heterologous protein in filamentous fungi.

Alcohol Dehydrogenase↗

PubMed1999

Unique DNA binding specificity of the binuclear zinc AlcR activator of the ethanol utilization pathway in Aspergillus nidulans.

AlcR is the transcriptional activator in Aspergillus nidulans, necessary for the induction of the alc gene cluster. It belongs to the Zn2Cys6 zinc cluster protein family, but contains some striking differences compared with other proteins of this group. In this report, we show that no dimerization element is present in the entire AlcR protein which occurs in solution as a monomer and binds also to its cognate sites as a monomer. Another important feature of AlcR is its unique specificity for single sites occurring naturally as inverted or direct repeats and sharing a common motif, 5'-(T/A)GCGG-3'. Like most other Zn2Cys6 proteins, AlcR contacts directly with the CGG triplet and, in addition, the upstream adjacent guanine is required for high affinity binding. We also establish that the flanking regions outside the core play an essential role in tight binding. From our in vitro analysis, we propose an optimal AlcR-binding site which is 5'-PuNGCGG-AT rich 3'.

Aspergillus nidulans↗

PubMed1999

A single amino acid, outside the AlcR zinc binuclear cluster, is involved in DNA binding and in transcriptional regulation of the alc genes in Aspergillus nidulans.

In Aspergillus nidulans, the transcriptional activator AlcR mediates specific induction of a number of alc genes. The AlcR DNA-binding domain is a zinc binuclear cluster that differs from the other members of the Zn2Cys6 family in several respects. Of these, the most remarkable is its ability to bind in vitro as a monomer to single sites, whereas only repeated sites (direct or inverted) are necessary and functional in vivo. Deletion of the first five amino acids (following the N-terminal methionine) upstream of the AlcR zinc cluster or mutation of a single residue, Arg-6, impairs the AlcR in vitro binding mainly to symmetrical sites. In vivo, the same mutations result in the inability of A. nidulans to grow on ethanol. The alc- phenotype results from a drastic decrease in activation of its own transcription and, in addition, that of the two structural genes, alcA and aldA, required for ethanol oxidation. This defect seems to be correlated to the inability of the Arg-6 AlcR mutant protein to bind to AlcR palindrome targets, which are essential in the three alc promoters. AlcR shows a unique pattern of binding and of transactivation among the Zn2Cys6 family.

Arginine↗

PubMed1997

In vitro recognition of specific DNA targets by AlcR, a zinc binuclear cluster activator different from the other proteins of this class.

AlcR is the transactivator mediating transcriptional induction of the alc gene cluster in Aspergillus nidulans. The AlcR DNA-binding domain consists of a zinc binuclear cluster different from the other members of the Zn2Cys6 family by several features. In particular, it is able to bind to symmetric and asymmetric sites with the same affinity, with both sites being functional in A. nidulans. Here, we show that unlike the other proteins of the Zn2Cys6 binuclear cluster family, AlcR binds most probably as a monomer to its cognate targets. Two molecules of the AlcR protein can simultaneously bind in a noncooperative manner to inverted repeats. The consensus core has been determined precisely (5'-CCGCN-3'), and the AlcR-binding site in the aldA promoter has been localized. The sequence downstream of the zinc cluster is necessary for high affinity binding. Furthermore, our data show that the use of the carrier protein glutathione S-transferase in AlcR binding experiments introduces an important bias in the recognition of DNA sites due to its tertiary dimeric structure.

Amino Acid Sequence↗

PubMed1996

Gene expression from replicating plasmids in Aspergillus nidulans.

Plasmids bearing the AMA1 replicator from Aspergillus nidulans are capable of extrachromosomal replication in this fungus as well as in other species. Synthetic plasmids bearing the moderately expressed argB gene and the highly expressed, inducible beta-galactosidase gene (bgaS) were introduced into fungal cells. Expression of both genes was monitored by Northern hybridization. It was demonstrated that transcription of bgaS is induced and repressed normally, irrespective of whether the gene is integrated into the chromosome or maintained on an extrachromosomal supercoiled plasmid. Transcription of the strongly expressed bgaS gene stimulates transcription of the argB gene located on the same replicating plasmid irrespective of orientation. This effect also occurs with chromosomally integrated vectors, but to a lesser extent. Episomal vectors are present in 10 copies per nucleus, and the expression level of the argB gene is approximately proportional to copy number. However, the amount of mRNA transcribed from the highly expressed bgaS gene on the multi-copy replicating plasmid does not exceed that from single-copy integrants. High levels of expression of the plasmid-borne gene do not affect plasmid mitotic stability or copy number.

Aspergillus nidulans↗