Production of extracellular proteins by the filamentous fungus Aspergillus.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
R F van Gorcom
Publications and source records attributed to R F van Gorcom.
Genetic analysis of Aspergillus niger mutants defective in benzoate-4-hydroxylase function.
This study was prompted by the observation that an Aspergillus niger transformant with a multicopy bphA (benzoate-4-hydroxylase gene) insert did not grow on benzoate, whereas a transformant with only one extra copy could grow. Therefore, an extensive survey has been made for other genes involved in the conversion of benzoate into 4-hydroxy-benzoate. A transformant with two copies of the bphA gene was used in part of the mutation experiments in order to avoid the isolation of many bphA mutants. Filtration enrichment was used to isolate mutants defective in the conversion of benzoate. The Bph mutants that have been isolated belong to six complementation groups. Mutants with a defected structural gene (bphA) were again predominantly found but, in addition, five other groups of mutants that could not grow on benzoate were isolated. Genetic analysis of the mutants showed that the six genes were localized in different parts of the genome. This was used as an additional proof that some mutants involved different genes. Diploids with seven copies of the bphA gene and heterozygous for one of the other bph genes were constructed. No indication has been obtained that any one of the mutant classes is responsible for the growth-limiting factor in bphA multicopy transformants. This study shows that the p-hydroxylation of benzoate is very complex, although the metabolic pathway is straight forward.
Aspergillus niger↗
Cloning of the nitrate-nitrite reductase gene cluster of Penicillium chrysogenum and use of the niaD gene as a homologous selection marker.
A new homologous transformation system for the filamentous fungus Penicillium chrysogenum is described. The system is based on complementation of niaD mutants using the nitrate reductase structural gene (niaD) of P. chrysogenum. Spontaneous niaD mutants were identified after selection for chlorate resistance, in growth tests and subsequent complementation with the niaD gene of Aspergillus oryzae. The P. chrysogenum niaD gene was isolated from a genomic library using the Aspergillus nidulans niaD gene as a probe. After subcloning of the hybridizing fragment, the vector obtained, pPC1-1, was capable of transforming a P. chrysogenum niaD mutant at an average of 40 transformants per micrograms of circular DNA. Southern analysis of genomic DNA from a number of transformants showed that pPC1-1 DNA was integrated predominantly at sites other than the niaD locus. Using hybridization analysis it was shown that the niaD gene of P. chrysogenum is clustered with the nitrite reductase gene (niiA). From analysis of the nucleotide sequences of parts of the niaD and niiA genes of P. chrysogenum and comparison of these sequences with nucleotide sequences of the corresponding A. nidulans genes it was deduced that the P. chrysogenum genes are divergently transcribed.
Cloning, Molecular↗
Isolation and molecular characterisation of the benzoate-para-hydroxylase gene (bphA) of Aspergillus niger: a member of a new gene family of the cytochrome P450 superfamily.
The gene coding for benzoate-para-hydroxylase (bphA) of Aspergillus niger was cloned using differential hybridisation techniques and complementation of mutants deficient in this enzyme activity. The nucleotide sequence of the gene was determined, the presence of two introns was shown and the transcription start and termination sites were determined. The structure of the mRNA upstream from the long open reading frame (ORF) is unusual. It contains two small, overlapping ORFs whose function is unknown. Comparison of the deduced amino acid sequence of the protein with the sequences present in the databanks, indicated a significant similarity of BPH to the superfamily of cytochrome P450 enzymes. Further analysis revealed that this protein is a member of a new P450 gene family designated P450LIII. The gene is designated CYP53. To increase the BPH activity of A. niger, multiple copies of the bphA gene were introduced into the genome of a recipient strain by transformation. Although increased intracellular levels of the BPH protein could be detected, the BPH enzyme activity was decreased, suggesting titration of another essential component.
Amino Acid Sequence↗
Expression analysis vectors for Aspergillus niger.
Explore the source record for details and available documents.
Aspergillus niger↗
A system for the analysis of expression signals in Aspergillus.
To analyse gene expression signals in Aspergillus we have constructed a set of integration vectors each of which contains in front of the Escherichia coli 'lacZ gene a unique BamHI site in one of the three possible translational reading frames and the A. nidulans argB gene as a selection marker. The vectors allow in-phase translational fusion of any gene to 'lacZ. After transformation of an A. nidulans argB strain, the vectors integrate with a high percentage at the argB locus of the genome, as a single copy. The insertion of the fusion genes at the argB locus assures the constancy of influences of the chromosomal environment on gene expression.
Aspergillus nidulans↗
Gene amplification in Aspergillus nidulans by transformation with vectors containing the amdS gene.
Conidial protoplasts of an A. nidulans amdS deletion strain (MH1277) have been transformed to the AmdS+ phenotype with a plasmid carrying the wild type gene (p3SR2). Optimalisation of transformation and plating conditions now has resulted in frequencies of 300-400 transformants per microgram of DNA. Analysis of DNA from AmdS+ transformants of MH1277 showed that transformation had occurred by integration of vector DNA sequences into the genome. In virtually all these transformants multiple copies of the vector were present in a tandemly repeated fashion, not preferentially at the resident, partially deleted amdS gene. It is suggested that the observed integration phenomena are dependent on the genetic background of the A. nidulans strain, used for transformation. A model to explain the tandem type of integration is proposed.
Aspergillus nidulans↗
Expression of an Escherichia coli beta-galactosidase fusion gene in Aspergillus nidulans.
We inserted in frame the Escherichia coli lacZ gene into the protein-coding region of the Aspergillus nidulans trpC gene and introduced the resultant fused gene into the A. nidulans genome. A functional beta Gal fusion protein was produced. Removal of the trpC transcription and translation initiation sequences from the fusion gene abolished production of the fusion protein, showing that expression is dependent on these sequences. Thus, lacZ fusions should be of use for estimating gene activity in a. nidulans.
Aspergillus nidulans↗