DNA-mediated transformation of the filamentous fungus Aspergillus nidulans

Although transformation of S. cerevisiae and N.crassa already could be achieved at the end of the seventies, positive results for A.nidulans had to await the isolation of useful selection markers. As soon as cloned fungal genes of homologous ( amd S, trp C and arg B from A.nidulans ) and heterologous ( pyr 4 from N.crassa ) origin became available transformation procedures for A.nidulans were developed (Ballance et al. 1983; Tilburn et al. 1983; Yelton et al. 1984; John and Pederby 1984). They all are based on the ability of these selection markers to complement auxotrophic A.nidulans mutants.A disadvantage of these transformation markers is the need for an auxotrophic recipient strain. With dominant selection markers even wild type strains should be good recipients for transformation, However, dominant selection markers like bacterial drug resistance genes, could not be developed due to the insensitivity of A.nidulans for most antibiotics (chapter 2). As found later in our studies in some conditions the amd S gene may serve as a dominant selection marker. All A.nidulans transformation protocols originate from that or S.cerevisiae , being based on the incubation of protoplasts with DNA in the presence of CaCl 2 and polyethylene glycol (PEG).In our study on A.nidulans transformation we initially focussed on the amd S marker (chapter 2, 3 and 4). Transformation of AmdS -strains with vectors containing the wild type amdS gene gives rise to two types of transformant colonies, viz. well growing, sporulating ones (type I) and tiny non-sporulating ones, with stagnating growth (type II). This latter type is not specific for the amd S marker, since with variable frequencies these have also been observed with other transformation markers (Yelton et al. 1984; John and Peberdy 1984; Ballance and Turner 1985; chapter 6). In general, these colonies have been indicated as "abortives". This, however is not correct since at least 50% of the type II AmdS +transformant colonies can be converted into type 1 (chapter 2).All type I AmdS +transformants, obtained with amd S containing vectors have integrated the transforming vector DNA sequences into the fungal genome DNA, as could be shown by Southern blotting analysis (chapter 2) and confirmed by genetic analysis (chapter 3). The integration of the transforming vector DNA into the genome is a common feature of the amd S gene and other cloned genes ( pyr 4, trp C, arg B), However. between the various selection markers, differences exist with respect to mode of vector DNA integration and transformation frequencies obtained (Ballance et al. 1983; Yelton et al. 1984; John and Peberdy 1984). The mode of amd S integration depends on the recipient A.nidulans AmdS -strain. Whereas strain WG290 usually integrates one single vector copy at the homologous, partially deleted amd S locus, virtually all AmdS +transformants of strain MH1277 contain multiple vector copies, integrated in tandemly repeated fashion. Integration is not preferentially at the homologous locus, nor at another specific site in the genome (chapter 2, chapter 3). Although integration of multiple vector copies into the A.nidulans genome has been observed using other selection markers, such a strain dependency has not been reported before. A model to explain the tandem type of integration in strain MH1277 (chapter 2) assumes the presence of a cryptic mutation in this acceptor. Such a locus has not been identified by genetic analysis. However, in diploid combinations of MH1277 derived AmdS +transformants and a master strain, unusually high levels of mitotic recombination are found (chapter 3). It is suggested that this is the basis of the peculiar mode of vector integration in MH1277.Genetic analysis of MH1277 derived AmdS +transformants confirms the conclusion derived from the biochemical analysis (chapter 2), that the transformant property is genome-linked; in six transformants analyzed the AmdS +property resides on five different chromomes. One of the transformants contains a translocation between two chromosomes of which at least one carries the AmdS +property. Translocation and vector integration in this strain may have occurred as two unrelated events. On the other hand it can be speculated that the former is a result of the latter.In chapter 4 a study is presented concerning the isolation of transforming vector sequences from the DNA of MR1277-derived AmdS +transformants via E.+coli . Digestion of the A.nidulans DNA with Eco RI, followed by ligation prior to E.coli transformation, yields plasmids even from a strain carrying only one single integrated vector copy. Following this procedure with AmdS +transformants containing multiple copy vector inserts, plasmid molecules can be recloned at higher frequencies. The length polymorphism found among these plasmids probably reflects the sequence rearrangements within the tandem inserts (chapter 2) and the recloning frequency shows a correlation with the number of vector copies integrated in each A.nidulans transformant.Similar vector plasmids could also be reisolated from undigested AmdS +transformant DNA. CsCl/EtBr centrifugations clearly demonstrate the presence of free covalently closed circular plasmid molecules within these A.+nidulans DNA preparation. Our opinion is that these plasmids arise invivo from recombination events between the individual copies within then tandem vector inserts, which are present in the genomic DNA of MH1277-derived AmdS +transformants. Also for A.nidulans transformants, obtained with other selection markers indications have been found for the presence of free vector molecules. Although some favour the idea of autonomous vector replication (Barnes and McDonald 1986) we consider this possibility unlikely.Chapter 5 deals with the phenomenon of cotransformation. When amd S mutants of A.nidulans are transformed with a mixture of an amd S containing vector and another, unlinked DNA sequence, a large fraction of the AmdS +transformants also contains this second, unselected sequence (chapter 5). The cotransformation frequency is demonstrated to depend both on the molar ratio of the two vectors and the concentration of the cotransforming vector. Although there may be some variation in the extent of cotransformation, it is in general such an efficient process in A.nidulans that the DNA of the unselected sequence can be found in almost every transformed cell.Cotransformation has been applied to induce gene replacement events in the A.nidulans genome (chapter 5). The amd S mutant WG290 was transformed with an amd S vector in the presence of a DNA fragment, containing an A.nidulanstrp C - E.coli lac Z (TrpC -, LacZ +) hybrid gene and among the AmdS +transformants we have screened for TrpC -. LacZ +colonies. Since tryptophan auxotrophs arise very infrequently. an enrichment procedure for TrpC -conidia has been applied to demonstrate the presence of the TrpC -transformants. We used ten such AmdS +, TrpC -transformants, which were all lacZ +, to study gene replacement. They were each transformed to TrpC +phenotype with a DNA fragment containing the wild type A.nidulanstrp C gene. Only 2 strains yielded at a low frequency, transformants which had simultaneously lost their LacZ +phenotype. These TrpC +, lacZ -colonies had the AmdS -phenotype. Southern blotting analysis of the two AmdS +, TrpC -, LacZ +mutants showed replacement of their wild type trp C gene by a trp C, lac Z, amd S-cointegrate. These results show that gene replacement by cotransformation is possible in A.nidulans , although less straight forward than directly selectable gene replacements (Miller et al. 1985). Due to the integrative behaviour of DNA sequences in A.nidulans , gene replacement procedures are more complex than in S.+cerevisiae ; In the latter case homologous recombination in the dominant mode of stable integration.In chapter 6 experiments are described in which the effect of the A.nidulansans l DNA fragment (Ballance and Turner 1985) on the frequency of Aspergillus transformation is examined, using the N.crassapyr 4 gene and the A.nidulans . amd S, arg B and trp C genes as selection markers. We find that ans l can increase transformation frequencies when added on a cotransforming vector with trp C, amd S and pyr 4, but not with arg B. When ans l is inserted into the vector, again with arg B no stimulation is found. In amd S vectors, the position of ans l with respect to the ans lS gene determined its influence on transformation: ans l upstream of amd S increased the frequency, whereas ans l downstream of amd S has no effect. Moreover the transformation frequency of the latter type of vector can not be stimulated by addition of ans l on a cotransforming vector. We suggest that ans l dependent stimulation involves an ans l gene product which, due to its inconsistency in effect may need a specific site for its action. The abolishing effect of DNA sequences like amd S may complicate the general applicability of this sequence in transformation.Transformation of A.nidulans has now evolved to a stage in which many problems can be tackled at a molecular level: cloning of genes in A.nidulans , introduction and expression of cloned genes, either from A.nidulans itself or from other organisms, study of the regulation of gene expression in A.nidulans using gene replacements, site directed mutagenesis etc. Moreover, the experience obtained with A.nidulans transformation can now be applied to other, biotechnologically important species like A.niger (see chapter 1).</TT

Molecular identification of ectomycorrhizal mycelium in soil horizons

Molecular identification techniques based on total DNA extraction provide a unique tool for identification of mycelium in soil. Using molecular identification techniques, the ectomycorrhizal (EM) fungal community under coniferous vegetation was analyzed. Soil samples were taken at different depths from four horizons of a podzol profile. A basidiomycete-specific primer pair (ITS1F-ITS4B) was used to amplify fungal internal transcribed spacer (ITS) sequences from total DNA extracts of the soil horizons. Amplified basidiomycete DNA was cloned and sequenced, and a selection of the obtained clones was analyzed phylogenetically. Based on sequence similarity, the fungal clone sequences were sorted into 25 different fungal groups, or operational taxonomic units (OTUs). Out of 25 basidiomycete OTUs, 7 OTUs showed high nucleotide homology (greater than or equal to99%) with known EM fungal sequences and 16 were found exclusively in the mineral soil. The taxonomic positions of six OTUs remained unclear. OTU sequences were compared to sequences from morphotyped EM root tips collected from the same sites. Of the 25 OTUs, 10 OTUs had greater than or equal to98% sequence similarity with these EM root tip sequences. The present study demonstrates the use of molecular techniques to identify EM hyphae in various soil types. This approach differs from the conventional method of EM root tip identification and provides a novel approach to examine EM fungal communities in soil

Tomato protoplast DNA transformation: physical linkage and recombination of exogenous DNA sequences

Tomato protoplasts have been transformed with plasmid DNA's, containing a chimeric kanamycin resistance gene and putative tomato origins of replication. A calcium phosphate-DNA mediated transformation procedure was employed in combination with either polyethylene glycol or polyvinyl alcohol. There were no indications that the tomato DNA inserts conferred autonomous replication on the plasmids. Instead, Southern blot hybridization analysis of seven kanamycin resistant calli revealed the presence of at least one kanamycin resistance locus per transformant integrated in the tomato nuclear DNA. Generally one to three truncated plasmid copies were found integrated into the tomato nuclear DNA, often physically linked to each other. For one transformant we have been able to use the bacterial ampicillin resistance marker of the vector plasmid pUC9 to 'rescue' a recombinant plasmid from the tomato genome. Analysis of the foreign sequences included in the rescued plasmid showed that integration had occurred in a non-repetitive DNA region. Calf-thymus DNA, used as a carrier in transformation procedure, was found to be covalently linked to plasmid DNA sequences in the genomic DNA of one transformant. A model is presented describing the fate of exogenously added DNA during the transformation of a plant cell. The results are discussed in reference to the possibility of isolating DNA sequences responsible for autonomous replication in tomato.

Occurrence of Escherichia coli O157 and other verocytotoxin-producing E.coli n retail raw meats in the Netherlands

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Survival and dispersal of a genetically modified Pseudomonas fluorescens strain during an experimental field release

In april 1995 werd door onderzoekers van het Instituut voor Plantenziektekundig Onderzoek (IPO/DLO) een kleinschalig veldexperiment gestart waarbij eenmalig een genetisch gemodificeerde Pseudomonas fluorescens stam (RIWE8) werd aangebracht in de toplaag van een proefveldje. In opdracht van het Ministerie van VROM werd door onderzoekers van het RIVM onderzoek uitgevoerd naar de overleving en verspreiding van het geintroduceerde genetisch gemodificeerde micro-organisme (ggm). Gedurende het eerste half jaar na beenting werden op gezette tijdstippen grondmonsters genomen voor analyse met microbiologische technieken. Hierbij werd het aantal kolonievormende deeltjes (cfp) bepaald van P. fluorescens RIWE in deze monsters, evenals het aantal van de ongemodificeerde ouderstam die op een naastgelegen controleveldje op identieke wijze was aangebracht. In de be-ente toplaag van het proefveldje werd een afname geconstateerd van de aantallen kolonievormende deeltjes van de geintroduceerde micro-organismen ter grootte van 4 tot 5 log10-eenheden. Reeds op dag 2 na be-enting werd verspreiding vanuit de toplaag waargenomen, zowel in de diepte als lateraal. Aanzienlijke aantallen micro-organismen werden waargenomen op een diepte van 20 - 25 cm en ook laterale verspreiding kon worden geconstateerd, zij het in geringere hoeveelheden. Aan het einde van de waarnemingsperiode konden de verspreide micro-organismen nog slechts op enkele monsterpunten en in lage aantallen worden aangetoond. Wat betreft overleving en verspreiding zijn er in dit onderzoek geen aanwijzingen gevonden voor verschillen tussen Pseudomonas fluorescens RIWE8 en de ongemodificeerde ouderstam.A small-scale field test was started in April 1995 by researchers of the Research Institute for Plant Protection (IPO/DLO) at Wageningen, the Netherlands), in which a genetically modified Pseudomonas fluorescens strain (RIWE8) was applied once to the top layer of a field plot. At request of the Dutch Ministry of Housing, Spatial Planning and Environment researchers of the RIVM carried out a study to monitor the survival and dispersal of the introduced, genetically modified micro-organism (gmm). During the first half-year period after inoculation soil samples were taken at fixed moments and analyzed by microbiological methods. The number of colony forming particles (cfp) in these samples was determined, as well as that of the non-modified parental strain which had been applied in an identical way to a neighbouring control plot. It was found that a 4 to 5 log10-units reduction occurred in the concentration of the introduced microorganisms. At day 2 after inoculation downward and lateral dispersal from the top layer was already observed. Considerable numbers of microorganisms were found at a depth of 20 - 25 cm in the field plot and, to a lower extent, lateral dispersal was observed as well. At the end of the monitoring period dispersed microorganisms could only be shown to be present at a few sampling sites and in low concentrations. With respect to survival and dispersal no differences were found between Pseudomonas fluorescens RIWE8 and the unmodified parental strain.DGM/SV