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).
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| Format: | Doctoral thesis biblioteca |
| Language: | English |
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| Subjects: | aspergillus, genetic engineering, recombinant dna, genetische modificatie, |
| Online Access: | https://research.wur.nl/en/publications/dna-mediated-transformation-of-the-filamentous-fungus-aspergillus |
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| Summary: | 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). |
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