Neomycin resistance as a dominantly selectable marker for transformation of the zygomycete Absidia glauca

Summary A plasmid (pAmN61) containing the NPT II structural gene (neomycin phosphotransferase) fused to the N-terminal region of a homologous actin gene was used for the transformation of Absidia glauca protoplasts. Neomycin resistant transformants could be selected for on complete medium containing 1.2 mg/ml neomycin sulfate. The physical presence of plasmid DNA in Absidia glauca was demonstrated by retransformation into Escherichia coli and by Southern blot analysis. No integration of plasmid DNA at either one of the two actin loci was observed; Southern blot experiments provide evidence that pAmN61 is autonomously replicated in Absidia glauca.     

Integrative transformation of a zygomycete,Absidia glauca,with vectors containing repetitive DNA

Summary Experimental evidence for integration of transformed DNA into the genome of Absidia glauca, a member of the fungal class of zygomycetes is presented. According to the limited knowledge on the molecular biology of these fungi, autonomous replication of transformed plasmids seems to be the preferential mode of DNA propagation. By inserting fragments of highly repetitive DNA elements into an autonomously replicating vector conferring neomycin resistance, we were able to obtain integrative transformation events. With such plasmids we observed stable mitotic propagation of a selective marker gene (NPT II under the control of a homologous actin promoter). Analysis of DNA from transformants in Southern type experiments, as well as restriction analysis of retransformants into Escherichia coli, provide evidence that integration of foreign DNA into the genome of Absidia glauca is possible. These transformation events are often associated with the appearance of mutant phenotypes.     

Transformation of Rhizopus niveus using a bacterial blasticidin S resistance gene as a dominant selectable marker

Summary Rhizopus niveus has been transformed to blasticidin S resistance by vectors containing the bacterial blasticidin S resistance gene under the control of a Rhizopus promoter. Southern analysis of the total DNA from transformants indicated that the introduced DNA was rearranged, and that one of the transformants harbored extrachromosomal plasmids with rearranged DNA. Using this transformation system, the introduction of pUBSR101, a plasmid carrying the Escherichia coli lacZ gene fused to the promoter and the N-terminal region of the R. niveus aspartic proteinase-II (RNAP-II) gene, resulted in an increase of -galactosidase activity in the cell extract, indicating expression of the lacZ fusion gene in R. niveus. This is the first report of a transformation system for filamentous fungi using the blasticidin S resistance gene as a dominant selectable marker.     

Transformation of Neurospora crassa with the trp-1 gene and the effect of host strain upon the fate of the transforming DNA

Summary Neurospora trp-1 ⁺ transformants, obtained by transforming a trp-1 inl strain with plasmid DNA containing the wild type trp1 ⁺ gene, were characterized by genetic and Southern blot analyses. The transforming trp-1 gene integrated at or near the resident site in all of the trp-1 ⁺ transformants obtained with circular DNA or DNA cut within the trp-1 coding region. The frequency of homologous integration decreased substantially when the donor DNA was cleaved outside the trp-1 coding region. The transformants were very stable mitotically and, in general, also showed meiotic stability. Analysis of trp-1 ⁺ transformants obtained with another recipient strain, trp-1 ⁺ ga-2 aro-9 inl, showed that homologous integration of donor DNA occurred in only 20% of the transformants, whether circular or linear DNA was used. Thus, the host strain employed for transformation appears to be a major factor in determining the fate of transforming DNA. Southern blot analysis of transformants showed that integration of the transforming DNA at the homologous site occurred by double crossover or gene conversion events rather than by insertion of the entire plasmid DNA. Multiple and apparently non functional integration events were observed in some transformants.     

Integrative and replicative genetic transformation of Aureobasidium pullulans

A hybrid selectable marker for transformation was constructed by placing the promoter (TEF1p) from the gene encoding the Aureobasidium pullulans translation elongation factor 1- (TEF1) adjacent to the 5 end of the Escherichia coli hygromycin B phosphotransferase gene (HPT). Plasmids containing this hybrid gene (TEF1p/HPT) transformed A. pullulans strain R106 to a hygromycin B-resistant (HmB^R) phenotype. A PCR-generated DNA fragment consisting of the TEF1p/HPT resistance marker flanked by 41 bp of homologous DNA has also been shown to transform A. pullulans to HmB^R. Linearized plasmid DNA consistently produced more transformants than circular plasmid DNA. Analyses of 23 HmB^R transformants revealed integration of the plasmid in only eight of these transformants. In two transformants, integration into the largest chromosome (VIII) resulted in an alteration of the molecular karyotype. In four other transformants, integration occurred in chromosome VI (the chromosome containing TEF1) but only one was the result of homologous recombination with the genomic copy of the TEF1 promoter. The remainder of the transformants contained replicative plasmids that could be visualized on an agarose gel by ethidium bromide staining. These plasmids were generally 7–8 kb in size. One transformant appeared to contain four plasmids ranging in size from 4 to 8 kb, suggesting rearrangement of the transforming DNA. One plasmid obtained from a HmB^R A. pullulans transformant was able to transform E. coli to ampicillin resistance. However, after recovery from E. coli, this plasmid (approximately 4 kb) was unable to transform A. pullulans to HmB^R.     

Genetic analysis of transformation in a microconidiating strain of Neurospora crassa

Summary We have characterized Neurospora crassa transformants obtained with plasmid pDV1001 bearing the cloned catabolic dehydroquinase (qa-2 ⁺) gene (Hughes et al. 1983) and fluffy 268 host strain producing only uninucleate microconidia allowing to isolate individual transformation products. The percentage of transformed nuclei in the mycelium and their stability were determined by genetic analysis of microconidia produced on selective or non-selective medium. About half of the transformants originating from mycelial spheroplasts were apparently homokaryotic. Catabolic dehydroquinase activity was in agreement with the proportion of transformed nuclei. The DNAs from four transformants analyzed by Southern hybridization showed restriction fragments expected for integration of pDV1001 into genomic DNA by non-homologous recombination. No plasmids could be rescued from the undigested DNAs of the transformants by transformation of E. coli. One transformant, 8268-6, was unstable and generated a high proportion of segregants. Plasmid pDV1001 sequences were absent in their DNA. Colonies originating from microconidia of strain fl268-6 on selective plates often lost the transformed character. These results suggest that instability in this transformant is due to the loss of integrated plasmid sequences during vegetative growth.     

Transformation of Penicillium chrysogenum using the Aspergillus nidulans amdS gene as a dominant selective marker

Summary The Aspergillus nidulans acetamidase gene (amdS) has been used to transform Penicillium chrysogenum at low frequency. Several transformants were tested and shown to be mitotically stable. Southern blot analysis indicated that transforming DNA had integrated into the chromosomal DNA, possibly at multiple sites.     

Plasmid recovery from transformants and the isolation of chromosomal DNA segments improving plasmid replication in Neurospora crassa

Summary The efficient recovery of plasmid DNA from Neurospora crassa transformants is described. Lithium acetate-treated spores were transformed with plasmid DNA and grown in mass in liquid culture. The resulting mycelial growth was harvested and plasmid DNA was extracted and used to transform E. coli to ampicillin resistance. Although at low frequency, routine recovery of plasmid pSD3 which carries the Neurospora qa-2 ⁺ gene and pBR322 sequences has been demonstrated. About 10% of the recovered plasmids carried deletions and transformed Neurospora at a higher frequency. The liquid culture procedure was also used in attempts to isolate autonomously replicating sequences (ars). In order to select for a stable vector which contains an ars sequence, a clone bank containing a selectable marker (qa-2 ⁺) and Neurospora chromosomal BamHI fragments was constructed and used to transform Neurospora. Several plasmid isolates resulting from a screening of the clone bank showed an improvement in the efficiency of recovery from Neurospora transformants. The properties of one such isolated plasmid, pJP102, suggest that it may contain an ars sequence. Some potential applications of these results for cloning in Neurospora and other filamentous fungi are discussed.     

Transformation ofNeurospora crassa with circular and linear DNA and analysis of the fate of the transforming DNA

Summary We have conducted a detailed study of 108qa-2 ⁺ Neurospora transformants which were obtained by use of circular plasmid DNAs and various linear DNAs. Parallel genetic and molecular analyses have revealed that three classes of transformants can be identified: linked transformants, in which theqa-2 gene has integrated at the resident locus, unlinked transformants, where integration has occurred at other genomic sites, and a third class designated non-transmissible which fail to pass theqa-2 gene through a cross. The non-transmissible class comprises the majority of transformants and may identify those which harbor autonomously replicating plasmids. Evidence is presented which suggests that a 1.2 kB BamHI-Bg1IIqa-2 ⁺ DNA fragment might possess anars sequence. Transformation with linear plasmid DNAs and DNA fragments carrying theqa-2 gene resulted in a demonstrable increase in transformation frequency beyond that achieved with circular plasmid DNAs, but did not permit precise targeting to the resident locus. Southern analysis showed that linked transformants have only the normal residentqa-2 band whereas the unlinked transformants always possess the resident band plus at least one additional band. Multiple integration events appear to be common and include cases where only a portion of the transforming DNA has been integrated.     

The transformation of protoplasts of Leptosphaeria maculans to hygromycin B resistance

Summary Conditions are described for the efficient isolation and regeneration of protoplasts of a fungal pathogen of brassicas, Leptosphaeria maculans. Treatment of the protoplasts with DNA of the plasmid pAN7-1 (containing an E. coli hygromycin phosphotransferase gene with Aspergillus nidulans expression signals) and plating under selective conditions resulted in the formation of hygromycin 13-resistant colonies. Southern blot analysis of resistant colonies indicated that single copies of the plasmid had integrated into different sites in the genome. In twelve of the transformants analysed so far, the integration is stable through mitosis. The demonstration of efficient transformation is an essential first step in the molecular analysis of pathogenicity of this commercially important pathogen.     

Recovery of recombinant plasmids from Pleurotus ostreatus transformants

Summary A transformation system employing selectable resistance to hygromycin B has been developed for the mushroom-forming fungus, Pleurotus ostreatus. Vector pAN7-1, a commonly used non-replicative vector for integrative transformation in fungi, yielded 5–46 resistant colonies per g of DNA per 10⁷ viable protoplasts. Southern blot analysis of certain transformants revealed unexpected replicative plasmids containing pAN7-1 sequences, but modified for size, methylation and restriction enzyme pattern when compared to the initial transforming vector. Two such replicative derivatives of pAN7-1 have been rescued from P. ostreatus by cloning into Escherichia coli. Rescued plasmids have been used to probe DNA from untransformed P. ostreatus in an effort to identify fungal sequences that recombined in vivo with pAN7-1 to form replicative plasmids. Such replicative sequences have been localized in high molecular weight (chromosomal) DNA of wild-type P. ostreatus. Transformation has been obtained for P. ostreatus using a rescued plasmid, thereby confirming the role of this recombinant plasmid as a shuttle vector.     

DNA repair genes of Saccharomyces cerevisiae: complementing rad4 and rev2 mutations by plasmids which cannot be propagated in Escherichia coli

Summary The RAD4 gene of yeast required for the incision step of DNA excision repair and the REV2 (= RAD5) gene involved in mutagenic DNA repair could not be isolated from genomic libraries propagated in E. coli regardless of copy number of the shuttle vector in yeast. Transformants with plasmids conferring UV resistance to a rad4-4 or a rev2-1 mutant were only recovered if yeast was transformed directly without previous amplification of the gene bank in E. coli. DNA preparations from these yeast clones yielded no transformants in E. coli but retransformation of yeast was possible. This lead to the isolation of a defective derivative of the rad4 complementing plasmid. The modified plasmid was now capable of transforming E. coli but still interfered significantly with its growth.Dedicated to Prof. Dr. Fritz Kaudewitz on the occasion of his 65th birthday     

Characterization of inl + transformants of Neurospora crassa obtained with a recombinant cosmid-pool

Summary We constructed a Neurospora crassa gene library in a cosmid vector and used the cosmid-pool DNA to transform an inl, rg Neurospora crassa strain to inositol prototrophy. The inl ⁺ colonies obtained in this experiment proved to be integrative type transformants. Genetic analysis revealed that the integration event occurred at or near the inl locus. In one of the transformants the inl ⁺ trait exhibited mitotic and meiotic instability. In hybridization experiments free plasmids were detected in the F₁ progeny of the transformants. We were able to recover eleven different plasmids from the F₁ progeny of the transformants. None of these plasmids proved to carry a functional copy of the inl ⁺ gene as judged by its transforming ability. Possible explanations for the observed phenomena are discussed.     

Isolation of the DNA sequence coding indole-3-glycerol phosphate synthetase and Phosphoribosylanthranilate isomerase of Schizophyllum commune

Summary A Schizophyllum gene library was made in plasmid pRK9, Plasmids from this library were tested for their ability to complement several auxotrophic mutations of Escherichia coli. The goal was to isolate a Schizophyllum auxotrophic gene that could be used to transform a corresponding Schizophyllum auxotrophic mutant to prototrophy. Complementation was observed only for E. coli trpC indole 3-glycerol phosphate synthetase (IGPS) and phosphoribosyl-anthranilate isomerase (PRAI) mutations. Plasmids with a Schizophyllum sequence coding for both IGPS and PRAI activities were recovered from E. coli transformants. Expression of the Schizophyllum gene (TRP1) in E. coli is probably dependent on the Serratia marcescens promoter of plasmid pRK9. The DNA sequence containing the Schizophyllum TRP1 gene was not obviously rearranged in cloning.     

DNA-mediated transformation of the secondarily homothallic basidiomycete Coprinus bilanatus

Summary The Coprinus cinereus trp-2 gene which encodes a trifunctional protein of the tryptophan biosynthetic pathway was used to transform a trp-2 mutant of the secondarily homothallic species Coprinus bilanatus. Southern blot analysis confirmed the presence of transforming DNA in the genome of transformants and indicated the presence of tandemly duplicated copies of the plasmid in some of these. Although these two species of Coprinus are regarded as closely related, no cross-hybridisation between the homologous trp-2 genes was detected.     

Multiple-copy integration in the yeast Yarrowia lipolytica

Using an EcoRI-BglII fragment of the G unit of the rDNA of Y. lipolytica and a set of 11 deletions in the URA3 promoter, we have constructed several plasmids to test gene amplification in the rDNA. These plasmids contain the rDNA fragment for integration, defective versions of the URA3 gene, the XPR2 gene encoding alkaline extracellular protease (AEP) as a reporter gene, and part of the pBR322 plasmid for selection and replication in E. coli. Among these plasmids, one corresponds to a deletion which allows multiple integration into the rDNA (plasmid pINA773). Two other plasmids (pINA767 and pINA772) give multiple integration only with a mutated URA3 gene. Transformants carrying these three plasmids were tested for copy number, stability, chromosomal localization and AEP secretion. Transformants containing plasmids pINA767, 772 and 773 displayed an average copy number of 5, 12 and 25–60 copies respectively of the plasmid, as estimated by PCR and DNA hybridization. Integrations occurred in only one chromosome except for transformants containing 60 copies where copies were observed at least in two different chromosomes. Multiple integrations were found both as tandem repeats and as dispersed copies. Plasmid copy number was stable in both minimum and rich media, for strains containing less than ten copies per cells. However, for higher copy number, multiple integrations were stable only when AEP synthesis was not induced, while in inducing medium stability of the multiple integrations was dramatically affected.     

Transfer of genetic information from the mycoparasite Parasitella parasitica to its host Absidia glauca

The infection of the model organism Absidia glauca by P. parasitica is accompanied by the fusion of both mycelia. By two lines of evidence we were able to show that this process is associated with the transfer of genes. First, auxotrophically labelled A. glauca mutants are efficiently complemented as a consequence of transfer of the parasite's genetic material. Second, for a plasmidcoded dominant marker (neomycin resistance), which is expressed in either organism, we proved the presence of plasmid DNA in recombinant recipients by molecular analysis at the DNA level. We propose the term para-recombinants for describing recombinant inter-generic chimærae, which are generated as a consequence of mycoparasitism.     

Shuttle vectors for <Emphasis Type="Italic">Candida albicans</Emphasis>: control of plasmid copy number and elevated expression of cloned genes

Plasmids containing the inosine monophosphate dehydrogenase gene CaIMH3 from Candida albicans strain ATCC 32354 transform their host to resistance against mycophenolic acid (MPA). The transformants maintain the plasmids at a high copy number (20–40 per cell) and express the CaIMH3 gene at very high levels relative to untransformed controls. The plasmid copy number can be controlled by the concentration of MPA in the media. The transformation procedure is reproducible and the efficiency of transformation is high, up to 15,000 per microgram. Unrearranged plasmids are readily recovered by transforming total DNA from transformants back into Escherichia coli. C. albicans genes cloned into the plasmid are expressed at elevated levels relative to untransformed controls. A derivative vector containing the CaMAL2 promoter and termination sequences expresses the CaERG11 ORF at high levels and confers moderate resistance to fluconazole. These shuttle vectors should facilitate global genomics approaches in C. albicans that have been hampered by its diploid genome.     

The isolation of specific genes from the basidiomycete Schizophyllum commune

Summary We have developed a routine way to isolate genes directly from the basidiomycete fungus, Schizophyllum commune. Plasmid DNA from a genomic gene library was used to isolate five specific genes by complementation of Schizophyllum mutations via transformation. The mutant strains were deficient in the ability to synthesize either adenine (ade2 and ade5), uracil (ural, encoding orotidine-5-phosphate decarboxylase; OMPdecase), tryptophan (rpl, encoding indole-3-glycerol phosphate synthetase; IGPS) or para aminobenzoic acid (pab1). In each case, Southern analysis revealed that transformation to prototrophy was concomitant with the integration of vector sequence into the genome of the S. commune mutant. Total DNA from transformants was restricted, religated, and used to transform E. coli. Ampicillin resistant plasmids were recovered from E. coli and tested for their ability to transform the corresponding mutant of S. commune. Plasmids complementing the ade2, adeS, pabl, trpl, and ural mutations were recovered.