Aspergillus DNA Research Articles

Identification of Aspergillus brlA response elements (BREs) by genetic selection in yeast.

The brlA gene of Aspergillus nidulans plays a central role in controlling conidiophore development. To test the hypothesis that brlA encodes a transcriptional regulator and to identify sites of interaction for the BrlA polypeptide, we expressed brlA in Saccharomyces cerevisiae (yeast) strains containing Aspergillus DNA sequences inserted upstream of a minimal yeast promoter fused to the Escherichia coli lacZ gene. Initially, a DNA fragment from the promoter region of the developmentally regulated rodA gene was tested and shown to mediate brlA-dependent transcriptional activation. Two additional DNA fragments were selected from an Aspergillus genomic library by their ability to respond to brlA in yeast. These fragments contained multiple copies of a sequence motif present in the rodA fragment, which we propose to be sites for BrlA interaction and designate brlA response elements (BREs). DNA fragments containing BREs upstream of a minimal Aspergillus promoter were capable of conferring developmental regulation in Aspergillus. Deletion of BREs from the upstream region of rodA greatly decreased its developmental induction. Multiple copies of a synthetic oligonucleotide with the consensus sequence identified among the BREs mediated brlA-dependent transcriptional activation in yeast. The results show that a primary activity of brlA is transcriptional activation and tentatively identify sites of interaction for the BrlA polypeptide.

DNA topoisomerase I from mycelia of Aspergillus oryzae: Identification of a catalytic molecule in aggregated complex

DNA topoisomerase I was extracted from mycelia of the mould, Aspergillus oryzae. Aspergillus DNA topoisomerase I exhibited maximum activity at pH 6.0 in 20 mM phosphate buffer. The activity was stimulated ca 20-fold in the presence of 10 mM Mg2+, and addition of ATP had no effect on the activity. DNA-intercalator such as ethidium bromide did not inhibit the enzyme. Km values were determined as 2.9 × 10−6 M base pairs toward Col E1 DNA and 4.4 × 10−6 M base pairs toward pUC18 DNA. In the experiments using a partially purified enzyme, a 66 kDa protein was shown to bind tightly and irreversibly to DNA-cellulose, which was deduced to be a catalytic site of the enzyme. From the chromatographic behaviour in gel permeation, we speculate that the enzyme would form an aggregated complex in the native state.

Behaviour of recombinant plasmids in Aspergillus nidulans: structure and stability.

A pyrG- Aspergillus strain was transformed with plasmid pDJB-1, derived from pBR325 by insertion of the Neurospora crassa pyr4 gene (orotidine 5'-phosphate carboxylase), giving mitotically unstable transformants. Aspergillus DNA which acted as an "autonomously replicating sequence" (ARS) in yeast was inserted into pDJB-1 and the resulting construct, pDJB12.1, gave mitotically stable transformants when introduced into Aspergillus. Transformants obtained with pDJB-1 and pDJB12.1 gave few pyr- progeny in crosses to a pyrG+ strain. Southern hybridisation analysis of pyr+ transformants obtained with pDJB-1 revealed restriction fragments expected for integrated plasmid but transformants obtained with pDJB12-1 showed only bands derived from free plasmid. pDJB-1 and derivatives of pDJB12.1 could be recovered from transformants. These derivatives could not be explained by straightforward excision of integrated pDJB12.1 sequences but could result from recombination between plasmid molecules. Hybridisation of undigested transformant DNAs showed that the transforming DNA was present in a high molecular weight form. These results suggest: (1) pDJB12.1 derivatives and possibly pDJB-1 can replicate autonomously in Aspergillus; (2) A. nidulans DNA acting as an ARS in yeast enhances replication and/or segregation of transforming plasmids in Aspergillus; and (3) recombinant plasmids may undergo rearrangements when introduced into Aspergillus.

Cloning of Aspergillus niger genes in yeast. Expression of the gene coding Aspergillus β-glucosidase

The possibility of cloning filamentous fungal genes by expression in the yeast Saccharomyces cerevisiae has been studied. A genome bank of Aspergillus niger was made in E. coli using a yeast cosmid shuttle vector and over 10,000 different cosmid clones were individually isolated. Yeast transformants carrying Aspergillus DNA were screened for the expression of the genes for fungal secreted glycoproteins, β-galactosidase, β-glucosidase, and amyloglucosidase, and for the expression of fungal genes complementing yeast ura3 and leu2 mutations. Of the five Aspergillus genes studied, only one, β-glucosidase, was found to be expressed in yeast, and this at a low level. This suggests that there are essential differences between the genes of yeast and filamentous fungi.

Cloning and expression in Escherichia coli K-12 of the biosynthetic dehydroquinase function of the arom cluster gene from the eucaryote, Aspergillus nidulans

A 1.35 Md DNA HindIII fragment containing part of the arom gene cluster or cluster gene of Aspergillus nidulans encoding biosynthetic dehydroquinase (5-dehydroquinate hydrolyase) has been cloned in plasmid pBR322 on the basis of functional expression in Escherichia coli. The fungal fragment on pBR322, designated pHK29, complements a corresponding E. coli dehydroquinase structural gene (aroD) mutation. pHK29 contains one BamHI, HpaII, PstI, SmaI, XhoI and surprisingly, one HindIII site since pHK29 hybrid Aspergillus DNA is a HindIII fragment itself. The biosynthetic dehydroquinase activity extracted from E. coli strains, containing pHK29, had properties similar to those of the enzyme activity from Aspergillus. The protein specified by pHK29 appears to be 80 kd. No increase of dehydroquinase activity was found in polynucleotide phosphorylase deficient strains (pnp) of E. coli.