DNA-mediated transformation of the filamentous fungus Aspergillus nidulans

K. Wernars

Research output: Thesisinternal PhD, WU

Abstract

<p>Although transformation of <u>S.</u> <u>cerevisiae</u> and <u>N.</u><u>crassa</u> already could be achieved at the end of the seventies, positive results for <u>A.</u><u>nidulans</u> had to await the isolation of useful selection markers. As soon as cloned fungal genes of homologous ( <u>amd</u> S, <u>trp</u> C and <u>arg</u> B from <u>A.</u><u>nidulans</u> ) and heterologous ( <u>pyr</u> 4 from <u>N.</u><u>crassa</u> ) origin became available transformation procedures for <u>A.</u><u>nidulans</u> 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 <u>A.</u><u>nidulans</u> mutants.<p>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 <u>A.</u><u>nidulans</u> for most antibiotics (chapter 2). As found later in our studies in some conditions the <u>amd</u> S gene may serve as a dominant selection marker. All <u>A.</u><u>nidulans</u> transformation protocols originate from that or <u>S.</u><u>cerevisiae</u> , being based on the incubation of protoplasts with DNA in the presence of CaCl <sub><font size="-1">2</font></sub> and polyethylene glycol (PEG).<p>In our study on <u>A.</u><u>nidulans</u> transformation we initially focussed on the <u>amd</u> S marker (chapter 2, 3 and 4). Transformation of AmdS <sup>-</SUP>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 <u>amd</u> 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 <sup><font size="-1">+</font></SUP>transformant colonies can be converted into type 1 (chapter 2).<p>All type I AmdS <sup><font size="-1">+</font></SUP>transformants, obtained with <u>amd</u> 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 <u>amd</u> S gene and other cloned genes ( <u>pyr</u> 4, <u>trp</u> C, <u>arg</u> 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 <u>amd</u> S integration depends on the recipient <u>A.</u><u>nidulans</u> AmdS <sup>-</SUP>strain. Whereas strain WG290 usually integrates one single vector copy at the homologous, partially deleted <u>amd</u> S locus, virtually all AmdS <sup><font size="-1">+</font></SUP>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 <u>A.</u><u>nidulans</u> 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 <sup><font size="-1">+</font></SUP>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.<p>Genetic analysis of MH1277 derived AmdS <sup><font size="-1">+</font></SUP>transformants confirms the conclusion derived from the biochemical analysis (chapter 2), that the transformant property is genome-linked; in six transformants analyzed the AmdS <sup><font size="-1">+</font></SUP>property resides on five different chromomes. One of the transformants contains a translocation between two chromosomes of which at least one carries the AmdS <sup><font size="-1">+</font></SUP>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.<p>In chapter 4 a study is presented concerning the isolation of transforming vector sequences from the DNA of MR1277-derived AmdS <sup><font size="-1">+</font></SUP>transformants <u>via E.</u><sup><font size="-1">+</font></SUP><u>coli</u> . Digestion of the <u>A.</u><u>nidulans</u> DNA with <u>Eco</u> RI, followed by ligation prior to <u>E.</u><u>coli</u> transformation, yields plasmids even from a strain carrying only one single integrated vector copy. Following this procedure with AmdS <sup><font size="-1">+</font></SUP>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 <u>A.</u><u>nidulans</u> transformant.<p>Similar vector plasmids could also be reisolated from undigested AmdS <sup><font size="-1">+</font></SUP>transformant DNA. CsCl/EtBr centrifugations clearly demonstrate the presence of free covalently closed circular plasmid molecules within these <u>A.</u><sup><font size="-1">+</font></SUP><u>nidulans</u> DNA preparation. Our opinion is that these plasmids arise <u>in</u><u>vivo</u> from recombination events between the individual copies within then tandem vector inserts, which are present in the genomic DNA of MH1277-derived AmdS <sup><font size="-1">+</font></SUP>transformants. Also for <u>A.</u><u>nidulans</u> 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.<p>Chapter 5 deals with the phenomenon of cotransformation. When <u>amd</u> S mutants of <u>A.</u><u>nidulans</u> are transformed with a mixture of an <u>amd</u> S containing vector and another, unlinked DNA sequence, a large fraction of the AmdS <sup><font size="-1">+</font></SUP>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 <u>A.</u><u>nidulans</u> that the DNA of the unselected sequence can be found in almost every transformed cell.<p>Cotransformation has been applied to induce gene replacement events in the <u>A.</u><u>nidulans</u> genome (chapter 5). The <u>amd</u> S mutant WG290 was transformed with an <u>amd</u> S vector in the presence of a DNA fragment, containing an <u>A.</u><u>nidulans</u><u>trp</u> C - <u>E.</u><u>coli lac</u> Z (TrpC <sup>-</SUP>, LacZ <sup><font size="-1">+</font></SUP>) hybrid gene and among the AmdS <sup><font size="-1">+</font></SUP>transformants we have screened for TrpC <sup>-</SUP>. LacZ <sup><font size="-1">+</font></SUP>colonies. Since tryptophan auxotrophs arise very infrequently. an enrichment procedure for TrpC <sup>-</SUP>conidia has been applied to demonstrate the presence of the TrpC <sup>-</SUP>transformants. We used ten such AmdS <sup><font size="-1">+</font></SUP>, TrpC <sup>-</SUP>transformants, which were all lacZ <sup><font size="-1">+</font></SUP>, to study gene replacement. They were each transformed to TrpC <sup><font size="-1">+</font></SUP>phenotype with a DNA fragment containing the wild type <u>A.</u><u>nidulans</u><u>trp</u> C gene. Only 2 strains yielded at a low frequency, transformants which had simultaneously lost their LacZ <sup><font size="-1">+</font></SUP>phenotype. These TrpC <sup><font size="-1">+</font></SUP>, lacZ <sup>-</SUP>colonies had the AmdS <sup>-</SUP>phenotype. Southern blotting analysis of the two AmdS <sup><font size="-1">+</font></SUP>, TrpC <sup>-</SUP>, LacZ <sup><font size="-1">+</font></SUP>mutants showed replacement of their wild type <u>trp</u> C gene by a <u>trp</u> C, <u>lac</u> Z, <u>amd</u> S-cointegrate. These results show that gene replacement by cotransformation is possible in <u>A.</u><u>nidulans</u> , although less straight forward than directly selectable gene replacements (Miller et al. 1985). Due to the integrative behaviour of DNA sequences in <u>A.</u><u>nidulans</u> , gene replacement procedures are more complex than in <u>S.</u><sup><font size="-1">+</font></SUP><u>cerevisiae</u> ; In the latter case homologous recombination in the dominant mode of stable integration.<p>In chapter 6 experiments are described in which the effect of the <u>A.</u><u>nidulans</u><u>ans</u> l DNA fragment (Ballance and Turner 1985) on the frequency of Aspergillus transformation is examined, using the <u>N.</u><u>crassa</u><u>pyr</u> 4 gene and the <u>A.</u><u>nidulans</u> . <u>amd</u> S, <u>arg</u> B and <u>trp</u> C genes as selection markers. We find that <u>ans</u> l can increase transformation frequencies when added on a cotransforming vector with <u>trp</u> C, <u>amd</u> S and <u>pyr</u> 4, but not with <u>arg</u> B. When <u>ans</u> l is inserted into the vector, again with <u>arg</u> B no stimulation is found. In <u>amd</u> S vectors, the position of <u>ans</u> l with respect to the <u>ans</u> lS gene determined its influence on transformation: <u>ans</u> l upstream of <u>amd</u> S increased the frequency, whereas <u>ans</u> l downstream of <u>amd</u> S has no effect. Moreover the transformation frequency of the latter type of vector can not be stimulated by addition of <u>ans</u> l on a cotransforming vector. We suggest that <u>ans</u> l dependent stimulation involves an <u>ans</u> 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 <u>amd</u> S may complicate the general applicability of this sequence in transformation.<p>Transformation of <u>A.</u><u>nidulans</u> has now evolved to a stage in which many problems can be tackled at a molecular level: cloning of genes in <u>A.</u><u>nidulans</u> , introduction and expression of cloned genes, either from <u>A.</u><u>nidulans</u> itself or from other organisms, study of the regulation of gene expression in <u>A.</u><u>nidulans</u> using gene replacements, site directed mutagenesis etc. Moreover, the experience obtained with <u>A.</u><u>nidulans</u> transformation can now be applied to other, biotechnologically important species like <u>A.</u><u>niger</u> (see chapter 1).<p><TT></TT>
Original languageEnglish
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • van der Veen, J.H., Promotor, External person
  • van den Broek, H.W.J., Promotor
Award date2 Dec 1986
Place of PublicationWageningen
Publisher
Publication statusPublished - 1986

Keywords

  • aspergillus
  • genetic engineering
  • recombinant dna

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