Transformation of Aspergillus nidulans by the argB gene

Aspergillus nidulans argB mutant was transformed with the plasmid DNA containing the argB gene. Analysis of transformants revealed that transformation was due to integration of either argB gene alone or the whole plasmid DNA into the A. nidulans genome. In 5 out of 23 transformants studied, integration took place in the locus different than the original argB locus. The amplification of integrated sequences was often observed. Integrated DNA was found to be mitotically stable, while the meiotic stability depends on the mode of integration. The activity of the ornithine carbamoyltransferase (the argB gene product) was measured and in some transformants bearing the amplified argB sequence was found to be strongly elevated.     

Direct and indirect gene replacements in Aspergillus nidulans.

We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation. A linear DNA fragment containing a heteromorphic trpC+ allele was used to transform a trpC- strain to trpC+. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpC- allele in a minority of the strains. An A. nidulans trpC+ gene was inserted into the argB+ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC- argB+ strain to trpC+. Approximately 30% of the transformants were simultaneously argB-. The null argB allele had replaced the wild-type allele in a majority of these strains. The A. nidulans SpoC1 C1-C gene was modified by removal of an internal restriction fragment and introduced into a trpC- strain by transformation with a circular plasmid. A transformant containing a tandem duplication of the C1-C region separated by plasmid DNA was self-fertilized, and trpC- progeny were selected. All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified C1-C gene and lost the wild-type copy. Thus, it is possible with A. nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one- or two-step procedures similar to those developed for Saccharomyces cerevisiae.     

Transformation of a mycoparasitic Trichoderma harzianum strain with the argB gene of Aspergillus nidulans

A transformation system was developed for the mycoparasitic filamentous fungus Trichoderma harzianum, based upon complementation of auxotrophic mutants. Prototrophic transformants were obtained using plasmids pSal23 and pAN5-41B, carrying the Aspergillus nidulans argB gene, and both the Aspergillus nidulans argB and Escherichia coli lacZ genes, respectively.     

Transformation of Aspergillus niger using the argB gene of Aspergillus nidulans

A mutant of Aspergillus niger defective in ornithine transcarbamylase function was transformed with plasmids carrying a functional copy of the argB gene of Aspergillus nidulans after treatment of spheroplasts in the presence of polyethylene glycol and calcium ions. The plasmid pDG3 gave stable transformants at a frequency of 4 per microgram of input DNA. Southern blot analysis of DNA from transformants showed that pDG3 DNA had integrated into the A. niger chromosomes at a variety of locations. The transformants were phenotypically stable for many mitotic divisions. This procedure may potentially be used to insert any gene into the genome of A. niger. A cosmid shuttle vector, pDG1, for cloning in Aspergillus was also constructed.     

The importance of surface charge and hydrophobicity for the flocculation of chain-forming brewing yeast strains and resistance of these parameters to acid washing

Abstract This paper describes transformation of intact conidia of Aspergillus nidulans , auxotrophic for arginine, by using the biolistic process. The plasmid employed was pFB39, carrying the argB gene. The transformation frequency obtained was 81 transformants/ μg of DNA. Classical genetics and molecular analysis were conducted to analyse transformants and to determine in which chromosome integration took place.     

Sequence and centromere proximal location of a transformation enhancing fragment ans1 from Aspergillus nidulans.

The Aspergillus nidulans sequence ans1, previously known to enhance transformation frequencies of pyr4-based vectors, was shown to enhance the efficiency of argB and trpC-based vectors. Increased efficiencies could be obtained by constructing vectors containing argB and ans1 or by cotransforming selectable plasmids (containing argB, trpC, or pyr4) with the non-selectable ans1 sequence. The preponderance of evidence suggests that the mechanism of ans1 activity does not involve homologous recombination events, in spite of the presence of multiple regions of homology in the A. nidulans genome. Genetic mapping localized ans1 to the vicinity of the centromere of linkage group I. The nucleotide sequence of a 1.8 Kb functional subclone of ans1 was determined and found to be highly A + T rich (81%).     

Cloning an Aspergillus nidulans developmental gene by transformation.

We have developed a transformation system for Aspergillus nidulans giving a frequency of transformation high enough to screen a gene bank from which we were able to isolate and clone the A. nidulans developmental gene brlA by visual selection. The vector contains the selective marker argB+, and with it a frequency of transformation of 500 stable transformants/micrograms plasmid DNA can regularly be achieved. The evidence suggests that transformation is by integration but spontaneous excision of integrated plasmids is apparently frequent enough to allow the recovery of transforming plasmids in Escherichia coli.     

A versatile transformation system for the cellulolytic filamentous fungus Trichoderma reesei

An efficient transformation system for the cellulolytic filamentous fungus Trichoderma reesei has been developed. Transformation was obtained with plasmid carrying the dominant selectable marker amdS or the argB gene of Aspergillus nidulans, which was found to complement the respective argB mutation of T. reesei. The transformation frequency can be up to 600 transformants per microgram of transforming DNA. The efficiency of co-transformation with unselected DNA was high (approx. 80%). The transforming DNA was found to be integrated at several different locations, often in multiple tandem copies in the T. reesei genome. In addition, the Escherichia coli beta-galactosidase was expressed in T. reesei in enzymatically active form from the A. nidulans gpd promoter.     

Genetic analysis of Aspergillus nidulans unstable transformants obtained by the biolistic process.

Mitotically unstable Aspergillus nidulans argB+ transformants obtained by the biolistic process were studied in the present work. Hybridization signals from undigested DNA and pulsed-field chromosomal bands of the transformants suggested the introduced plasmid occurred as free concatenated molecules. Fifteen vigorous growth sectors released from the transformants were analysed in order to understand the mechanisms involved in their formation. All sectors showed the integration of exogenous genes into the fungal genome by homologous or heterologous recombinant events.     

An Aspergillus nidulans uvsC null mutant is deficient in homologous DNA integration

The Aspergillus nidulans uvsC gene was identified as a homolog of RAD51 and recA of Saccharomyces cerevisiae and Escherichia coli, respectively, whose role in genetic recombination and recombinational repair has been extensively studied. Like many other filamentous fungi, A. nidulans shows no bias towards either homologous or ectopic integration of exogenous DNA. Therefore it is a unique and useful organism for the study of the mechanisms of DNA integration. Homologous integration of a 1.7-kb argB gene was not detected in 50 transformants obtained from a uvsC null mutant. In contrast, the frequency of homologous integration in uvsC+ control strains varied from 41 to 86%. Another feature observed with the uvsC null mutant was that an increased number of transformants had undergone ectopic integrations at multiple sites in the genome. These results are consistent with the established function of Rad51/RecA, and further indicate the involvement of redundant pathways in integration of exogenous DNA. This study provides direct evidence for the involvement of uvsC in exogenous DNA integration and should contribute to the improvement of genetic manipulations in general, but particularly in fungi.     

Aspergillus nidulans verA is required for production of the mycotoxin sterigmatocystin.

Aspergillus nidulans produces the carcinogenic mycotoxin sterigmatocystin (ST), the next-to-last precursor in the aflatoxin (AF) biosynthetic pathway found in the closely related fungi Aspergillus flavus and Aspergillus parasiticus. We identified and characterized an A. nidulans gene, verA, that is required for converting the AF precursor versicolorin A to ST. verA is closely related to several polyketide biosynthetic genes involved in polyketide production in Streptomyces spp. and exhibits extended sequence similarity to A. parasiticus ver-1, a gene proposed to encode an enzyme involved in converting versicolorin A to ST. By performing a sequence analysis of the region 3' to verA, we identified two additional open reading frames, designated ORF1 and ORF2. ORF2 is closely related to a number of cytochrome P-450 monooxygenases, while ORF1 shares identity with the gamma subunit of translation elongation factor 1. Given that several steps in the ST-AF pathway may require monooxygenase activity and that AF biosynthetic genes are clustered in A. flavus and A. parasiticus, we suggest that verA may be part of a cluster of genes required for ST biosynthesis. We disrupted the verA coding region by inserting the A. nidulans argB gene into the center of the coding region and transformed an A. nidulans argB2 mutant to arginine prototrophy. Seven transformants that produced DNA patterns indicative of a verA disruption event were grown under ST-inducing conditions, and all of the transformants produced versicolorin A but negligible amounts of ST (200-fold to almost 1,000-fold less than the wild type), confirming the hypothesis that verA encodes an enzyme necessary for converting versicolorin A to ST.     

Apparent mRNA instability in Aspergillus nidulans and Aspergillus terreus of a heterologous cDNA encoding the major capsid antigen of Rotavirus

Two expression plasmids designed to produce the rotaviral VP6 protein in Aspergillus nidulans and Aspergillus terreus have been constructed. In one of these plasmids the inducible A. terreus Gla1 glucoamylase gene promoter and Gla1 signal sequence are fused to the VP6 cDNA to enable induction and extracellular secretion of the final protein product; in the other, the strong, constitutive A. nidulans gpdA gene promoter has been employed. A. nidulans and A. terreus transformants containing intact copies of these plasmids have been obtained but neither intra- nor extra-cellular VP6 protein was detectable. Northern analysis indicated specific degradation of the VP6 mRNA. This lack of VP6 mRNA stability may be related to fundamental differences between the general structure of Aspergillus mRNA and that of rotavirus, including codon usage and AU/GC ratio.     

Isolation and subcloning analysis of functional centromere DNA (CEN11) from Saccharomyces cerevisiae chromosome XI.

We have cloned segments of yeast DNA containing the centromere XI-linked MET14 gene. This was done by selecting directly in Saccharomyces cerevisiae for complementation of a met14 mutation after transformation with a hybrid plasmid DNA genomic library. Genetic evidence indicates that functional centromere DNA (CEN11) from chromosome XI is also contained on the segment of S. cerevisiae DNA cloned in pYe(MET14)2. This plasmid is maintained stably in budding S. cerevisiae cultures and segregates predominantly 2+:20- through meiosis. The CEN11 element has been subcloned in vector YRp7' on an S. cerevisiae DNA fragment 900 base pairs in length [pYe(CEN11)10]. The mitotic and meiotic behavior of plasmids containing CEN11 plus a DNA replicator (ars) indicates that the centromere DNA sequences enable these plasmids to function as true minichromosomes in S. cerevisiae.     

Analysis of Aspergillus niger transformants for single site integration and vector recombination

Summary In order to understand the mechanism of integrative transformation, bulk DNA from several transformants of Aspergillus niger was assaved for the presence of the heterologous vector p3SR2. In all cases vector sequences and recombinant vector fragments were found. Transformant T31-6 containing seven plasmid-probed bands was studied in detail. Recloning of six of these plasmids in Escherichia coli revealed that internal recombination events had produced deletions and insertions. Only one plasmid was isolated among 402 analysed clones that had involved A. niger DNA. Sequence analysis revealed that the integration of vector p3SR2 occurred via its A. nidulans amdS ⁺ sequence. These data support the assumption that the integration of multiple copies of p3SR2 occurred at a single genomic site.Offprint requests to: K. Esser     

Cloning the alpha-amylase gene of Bacillus amyloliquefaciens in cyanobacteria cells

The recombinant plasmids of pIAH4amy series were constructed containing the alpha-amylase gene of Bacillus amyloliquefaciens A50 with its own promoter and leading sequence within an integrative vector plasmid pIAH4 (CmR) for cyanobacterium Anacystis nidulans R2. At Anacystis nidulans transformation the hybrid plasmids integrate into cyanobacterium chromosome with high efficiency and all CmR transformants produce alpha-amylase. Expression of bacillar alpha-amylase gene in cyanobacterium cells is independent of the cloned gene orientation in the vector plasmid. Secretion of alpha-amylase into the cyanobacterial periplasm has been demonstrated.     

Identification of a telomeric fragment from the right arm of chromosome III of Aspergillus nidulans

A minimum of 11 bands hybridising to an oligonucleotide complementary to the putative telomeric repeat sequence (TTAGGG)n was visible in a Southern blot of EcoRI-digested Aspergillus nidulans genomic DNA. All 11 were sensitive to BAL 31 exonuclease digestion, consistent with telomeric locations. Blots of DNA from aneuploid strains deleted for a dispensable, extreme distal region on the right arm of chromosome III lack a 1.3-kb EcoRI band, indicating that this fragment is located at or near the chromosome III right arm telomere.