Molecular mechanisms of insertional mutagenesis in yeasts and mycelium fungi

Random insertional mutagenesis is an efficient tool for studying molecular mechanisms of many genetically determined processes. An improved variant of this method is REMI (Restriction Enzyme Mediated Integration) mutagenesis. In this method, the insertion cassette is introduced into the recipient cell together with restriction endonuclease. As a result, the REMI cassette insertion occurs in sites recognized by the restriction enzyme. The use of restriction endonucleases enhances transformation rate and provides cassette insertion in virtually any locus. A mutation is tagged by the insertion cassette, which can be identified by isolating the REMI cassette together with the flanking genomic DNA regions. The review describes general requirements to REMI. The mechanisms of REMI mutagenesis are surveyed with special reference to yeast Saccharomyces cerevisiae. Special attention is given to the development and use of REMI for other lower eukaryotes (yeasts and mould fungi). Drawbacks of the method and perspectives of its use are discussed.     

Mutagenesis via insertional- or restriction enzyme-mediated-integration (REMI) as a tool to tag pathogenicity related genes in plant pathogenic fungi.

Random insertional mutagenesis is a powerful tool to investigate the molecular basis of most genetically determined processes, for example in pathogenic fungi. An improved version of this method is the insertional mutagenesis via restriction enzyme mediated integration (REMI). Transformation efficiency and mode of vector integration are species dependent and further influenced by vector conformation, restriction enzyme activity, and transformation protocol. An overview is given, covering the mutants and already identified genes obtained after REMI mutagenesis. An outlook describes the future developments in the field.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants.     

Direct random insertion mutagenesis of Helicobacter pylori

Random insertion mutagenesis is a widely used technique for the identification of bacterial virulence genes. Most strategies for random mutagenesis involve cloning in Escherichia coli for passage of plasmids or for phenotypic selection. This can result in biased selection due to restriction or instability of the cloned DNA, or toxicity of the encoded products. We therefore created two mutant libraries in the human pathogen Helicobacter pylori using a simple, direct mutagenesis technique, which does not require E. coli as intermediate. H. pylori total DNA was digested, circularized and digested again with a frequently cutting restriction enzyme, and the resulting fragments were ligated to a kanamycin antibiotic resistance cassette. Subsequently, the ligation mixture was transformed into the parental H. pylori strain 1061. Insertion of the kanamycin cassette by double homologous recombination into the genome of H. pylori 1061 resulted in approximately 2500 kanamycin resistant colonies. Heterogeneity of kanamycin cassette insertion was confirmed by Southern blotting. The isolation of two independent H. pylori mutants defective in production of urease from this library underlines the potential of this mutagenesis strategy.     

Insertional mutagenesis of Aspergillus fumigatus

We have investigated transformation with heterologous DNA as a method for insertional mutagenesis of Aspergillus fumigatus. Two methods, polyethylene glycol-mediated transformation of protoplasts and electroporation of germinating spores, were used to establish conditions leading to single-copy integration of transforming DNA at different genomic sites. We have assessed the effect of restriction enzyme-mediated integration (REMI) for both methods. Non-REMI protoplast transformation led to integration of multiple copies of transforming DNA in the majority of transformants. Results of REMI with protoplast transformation varied depending on the enzyme used. Low concentrations of several restriction enzymes stimulated transformation, but of ten enzymes investigated only REMI with XhoI and KpnI resulted in single-copy integration of transforming DNA for the majority of transformants. For protoplast transformation with XhoI- or KpnI-based REMI, 50% and 76% of insertions, respectively, were due to integrations at a genomic enzyme site corresponding to the enzyme used for REMI. Electroporation of spores without addition of restriction enzyme resulted in a high transformation efficiency, with up to 67% of transformants containing a single copy of transforming DNA. In contrast to protoplast transformation, electroporation of spores in the presence of a restriction enzyme did not improve transformation efficiency or lead to insertion at genomic restriction sites. Southern analysis indicated that for both protoplast transformation with REMI using KpnI or XhoI and for electroporation of spores without addition of restriction enzymes, transforming DNA inserted at different genomic sites in a high proportion of transformants. Received: 6 March 1998 / Accepted: 25 May 1998     

Genetic analysis in fungi using restriction-enzyme-mediated integration

Restriction-enzyme-mediated integration (REMI), a method for generating nonhomologous integration of transforming DNA into the chromosomes of eukaryotic cells, has been used for insertion mutagenesis and other genetic studies in diverse organisms. Insertion mutations generated by REMI have facilitated the genetic dissection of developmental pathways in Dictyostelium discoidium and the isolation of virulence factors in several plant pathogenic fungi. Recent work indicates that REMI occurs by nonhomologous end joining.     

Cassette mutagenesis: an efficient method for generation of multiple mutations at defined sites

A method is described for the efficient insertion of mutagenic oligodeoxynucleotide cassettes which allow saturation of a target amino acid codon with multiple mutations. Restriction sites are introduced by oligonucleotide-directed mutagenesis procedures to flank closely the target codon in the plasmid containing the gene. The restriction sites to be introduced are chosen based on their uniqueness to the plasmid, proximity to the target codon and conservation of the final amino acid coding sequence. The flanking restriction sites in the plasmid are digested with the cognate restriction enzymes, and short synthetic duplex DNA cassettes (10-25 bp) are inserted. The mutagenic cassette is designed to restore fully the wild-type coding sequence, except over the target codon, and to eliminate one or both restriction sites. Elimination of a restriction site facilitates selection of clones containing the mutagenic oligodeoxynucleotide cassette. To make the cassettes, single-stranded oligodeoxynucleotides and their complements are synthesized in separate pools containing different codons over the target. This method has been successfully applied to generate 19 amino acid substitutions at position 222 in the subtilisin protein sequence.     

Tagging Hansenula polymorpha genes by random integration of linear DNA fragments (RALF)

We have investigated the feasibility of using gene tagging by restriction enzyme-mediated integration (REMI) to isolate mutants in Hansenula polymorpha. A plasmid that cannot replicate in H. polymorpha and contains a dominant zeocin resistance cassette, pREMI-Z, was used as the integrative/mutagenic plasmid. We observed that high transformation efficiency was primarily dependent on the use of linearised pREMI-Z, and that the addition of restriction endonuclease to linearised pREMI-Z prior to transformation increased the transformation frequency only slightly. Integration of linearised pREMI-Z occurred at random in the H. polymorpha genome. Therefore, we termed this method Random integration of Linear DNA Fragments (RALF). To explore the potential of RALF in H. polymorpha, we screened a collection of pREMI-Z transformants for mutants affected in peroxisome biogenesis (pex) or selective peroxisome degradation (pdd). Many previously described PEX genes were obtained from the mutant collection, as well as a number of new genes, including H. polymorpha PEX12 and genes whose function in peroxisome biogenesis is still unclear. These results demonstrate that RALF is a powerful tool for tagging genes in H. polymorpha that should make it possible to carry out genome-wide mutagenesis screens.     

piggyBac transposon-mediated transgenesis in the apicomplexan parasite Eimeria tenella

piggyBac, a type II transposon that is useful for efficient transgenesis and insertional mutagenesis, has been used for effective and stable transfection in a wide variety of organisms. In this study we investigate the potential use of the piggyBac transposon system for forward genetics studies in the apicomplexan parasite Eimeria tenella. Using the restriction enzyme-mediated integration (REMI) method, E. tenella sporozoites were electroporated with a donor plasmid containing the enhanced yellow fluorescent protein (EYFP) gene flanked by piggyBac inverted terminal repeats (ITRs), an Asc I-linearized helper plasmid containing the transposase gene and the restriction enzyme Asc I. Subsequently, electroporated sporozoites were inoculated into chickens via the cloacal route and transfected progeny oocysts expressing EYFP were sorted by flow cytometry. A transgenic E. tenella population was selected by successive in vivo passage. Southern-blotting analysis showed that exogenous DNA containing the EYFP gene was integrated into the parasite genome at a limited number of integration sites and that the inserted part of the donor plasmid was the fragment located between the 5' and 3' ITRs as indicated by primer-specific PCR screening. Genome walking revealed that the insertion sites were TTAA-specific, which is consistent with the transposition characteristics of piggyBac.     

Construction of linker-scanning mutations using a kanamycin-resistance cassette with multiple symmetric restriction sites

We demonstrate how a kanamycin-resistance (KmR) cassette flanked by polylinkers with multiple restriction sites can be used to introduce nucleotide (nt) sequence replacements into a region of interest. This method differs in two significant ways from traditional methods of linker mutagenesis. First, the presence of the KmR gene allows for selection of the polylinker, greatly facilitating formation of linker-containing molecules. Second, the polylinker with multiple restriction sites allows a given linker insertion to be combined with a second linker insertion in a variety of different ways and makes possible a range of novel nt to remain in the resulting linker replacement. The result of this flexibility is that fewer different molecules are needed to cover a region, and that relatively large replacements (greater than 40 nt) are possible. We have used this method to introduce a series of sequence replacements that span the mouse dihydrofolate reductase promoter region.     

Restriction enzyme-mediated integration used to produce pathogenicity mutants of Colletotrichum graminicola

We have developed a restriction enzyme-mediated insertional mutagenesis (REMI) system for the maize pathogen Colletotrichum graminicola. In this report, we demonstrate the utility of a REMI-based mutagenesis approach to identify novel pathogenicity genes. Use of REMI increased transformation efficiency by as much as 27-fold over transformations with linearized plasmid alone. Ninety-nine transformants were examined by Southern analysis, and 51% contained simple integrations consisting of one copy of the vector integrated at a single site in the genome. All appeared to have a plasmid integration at a unique site. Sequencing across the integration sites of six transformants demonstrated that in all cases the plasmid integration occurred at the corresponding restriction enzyme-recognition site. We used an in vitro bioassay to identify two pathogenicity mutants among 660 transformants. Genomic DNA flanking the plasmid integration sites was used to identify corresponding cosmids in a wild-type genomic library. The pathogenicity of one of the mutants was restored when it was transformed with the cosmids.     

The use of a selectable FokI cassette in DNA replacement mutagenesis of the R388 dihydrofolate reductase gene

FokI, a class-IIS restriction endonuclease, cleaves double-stranded DNA to produce a protruding 5' end consisting of four nucleotides, 10-13 residues 3' from the nonpalindromic recognition sequence, GGATG. Cassettes which utilize this separation of cleavage and recognition site have been constructed for the purpose of linker mutagenesis and DNA replacement experiments. The cassettes are flanked by FokI recognition sequences oriented such that the FokI cleavage sites are several nucleotides beyond the cassette/vector fusion sites. FokI excises the cassette and several base pairs of the neighboring vector sequence. The ends produced in the vector by FokI cleavage are generally noncomplementary and suitable for the insertion of a segment of synthesized double-stranded replacement DNA. A cassette which contains a tyrosine tRNA suppressor gene (supF) is selectable by the suppression of amber mutations in the recipient host. A vector containing a pBR322-derived origin of replication, the Escherichia coli xanthine-guanine phosphoribosyl transferase gene as a selectable marker, and no FokI sites has been constructed for use with the FokI cassettes. An experiment which utilized the FokI/supF cassette to modify the N-terminal coding region of the R388 dihydrofolate reductase gene is described.     

Codon cassette mutagenesis: a general method to insert or replace individual codons by using universal mutagenic cassettes.

We describe codon cassette mutagenesis, a simple method of mutagenesis that uses universal mutagenic cassettes to deposit single codons at specific sites in double-stranded DNA. A target molecule is first constructed that contains a blunt, double-strand break at the site targeted for mutagenesis. A double-stranded mutagenic codon cassette is then inserted at the target site. Each mutagenic codon cassette contains a three base pair direct terminal repeat and two head-to-head recognition sequences for the restriction endonuclease Sapl, an enzyme that cleaves outside of its recognition sequence. The intermediate molecule containing the mutagenic cassette is then digested with Sapl, thereby removing most of the mutagenic cassette, leaving only a three base cohesive overhang that is ligated to generate the final insertion or substitution mutation. A general method for constructing blunt-end target molecules suitable for this approach is also described. Because the mutagenic cassette is excised during this procedure and alters the target only by introducing the desired mutation, the same cassette can be used to introduce a particular codon at all target sites. Each cassette can deposit two different codons, depending on the orientation in which it is inserted into the target molecule. Therefore, a series of eleven cassettes is sufficient to insert all possible amino acids at any constructed target site. Thus codon cassettes are 'off-the-shelf' reagents, and this methodology should be a particularly useful and inexpensive approach for subjecting multiple different positions in a protein sequence to saturation mutagenesis.     

Using DNA-tagged mutagenesis to improve heterologous protein production in Aspergillus oryzae

Using DNA-tagged mutagenesis to improve heterologous protein production in Aspergillus oryzae. Fungal Genetics and Biology 29, 28-37. Restriction enzyme-mediated integration (REMI) has been employed as a mutagen to generate two insertion libraries in an Aspergillus oryzae strain expressing a Thermomyces lanuginosus lipase. The REMI libraries were created using linearized plasmid containing the A. oryzae pyrG and either BamHI or EcoRI enzyme. The libraries were screened for lipase production, and mutants with increased production were isolated. The genomic DNA flanking the integration event was cloned from one of the mutants with increased lipase titers (DEBY10.3). Nucleotide sequence of the flanking DNA revealed similarity to the Aspergillus nidulans palB gene. Disruption of the palB gene in a strain producing lipase resulted in increased lipase expression. Additionally, complementation of the palB phenotype of DEBY10.3 led to a decrease in lipase production. These lines of evidence demonstrate that the increase in lipase yield in DEBY10.3 is linked to the palB phenotype generated by the integration of the pyrG gene into the palB gene. The results also demonstrated that tagged mutagenesis with REMI can be used to identify genes that influence expression of heterologous proteins.     

Deletions in the gibberellin biosynthesis gene cluster of Gibberella fujikuroi by restriction enzyme-mediated integration and conventional transformation-mediated mutagenesis.

We induced mutants of Gibberella fujikuroi deficient in gibberellin (GA) biosynthesis by transformation-mediated mutagenesis with the vector pAN7-1. We recovered 24 GA-defective mutants in one of nine transformation experiments performed without the addition of a restriction enzyme. Each mutant had a similar Southern blot pattern, suggesting the integration of the vector into the same site. The addition of a restriction enzyme by restriction enzyme-mediated integration (REMI) significantly increased the transformation rate and the rate of single-copy integration events. Of 1,600 REMI transformants, two produced no GAs. Both mutants had multiple copies of the vector pAN7-1 and one had a Southern blot pattern similar to those of the 24 conventionally transformed GA-deficient mutants. Biochemical analysis of the two REMI mutants confirmed that they cannot produce ent-kaurene, the first specific intermediate of the GA pathway. Feeding the radioactively labelled precursors ent-kaurene and GA12-aldehyde followed by high-performance liquid chromatography and gas chromatography-mass spectrometry analysis showed that neither of these intermediates was converted to GAs in the mutants. Southern blot analysis and pulsed-field gel electrophoresis of the transformants using the bifunctional ent-copalyl diphosphate/ent-kaurene synthase gene (cps/ks) and the flanking regions as probes revealed a large deletion in the GA-deficient REMI transformants and in the GA-deficient transformants obtained by conventional insertional transformation. We conclude that transformation procedures with and without the addition of restriction enzymes can lead to insertion-mediated mutations and to deletions and chromosome translocations.     

Selection of an effective red-pigment producing Monascus pilosus by efficient transformation with aurintricarboxylic acid

The filamentous fungus Monascus pilosus was genetically transformed with a reporter plasmid, pMS-1.5hp, by aurintricarboxylic acid (ATA) treatment to obtain an efficient red-pigment producing mutant. The transformation efficiency of Monascus pilosus was higher with the ATA-treatment than with either a non-restriction-enzyme-mediated integration (REMI) or a REMI method. This valid and convenient random mutagenesis method shows that ATA can be applied in fungi for efficient genetic transformation.