Tagging pathogenicity genes inUstilago maydis by restriction enzyme-mediated integration (REMI)

In the maize pathogenic fungusUstilago maydis integration of transforming DNA at homologous or heterologous sites is often accompanied by duplications of the DNA. We show that it is possible to generate single-copy integration events with high efficiency by restriction enzyme-mediated integration (REMI). In about 50% of cases, a plasmid that contains a singleBamHI site is integrated at chromosomalBamHI sites, ifBamHI is added to the transformation mixtures. In the other cases it appears that integration events have also occurred preferentially atBamHI sites, but without restoration of the recognition sites. Using REMI we have generated approximately 1000 insertion mutants. Pathogenicity tests demonstrated that about 1–2% of these mutants were unable to induce symptoms when testedin planta. For two of the mutants we have shown that the phenotype is linked to the insertion event.     

Restriction enzyme-mediated DNA integration in Coprinus cinereus

Restriction enzyme-mediated DNA integration (REMI) has recently received attention as a new technique for the generation of mutants by transformation in fungi. Here we analyse this method in the basidiomycete Coprinus cinereus using the homologous pab1 gene as a selectable marker and the restriction enzymes BamHI, EcoRI and PstI. Addition of restriction enzymes to transformation mixtures results in an earlier appearance of transformants and influences transformation rates in an enzyme- and concentration-dependent manner. Low concentrations of restriction enzyme result in increased numbers of transformants compared to no addition of enzymes. Transformation rates decrease with higher enzyme concentrations. If protoplasts are made from cells stored in the cold, the transformation rates drop drastically even in the presence of low amounts of enzyme. In several transformants, plasmid integration directly correlated with the action of restriction enzyme at random chromosomal restriction sites. In some cases, restriction enzymes appear to reduce the number of integration events per transformant. Simultaneously, mutation rates can be enhanced due to the presence of restriction enzymes. Although restriction enzymes clearly promote plasmid integration into the host genome they also have cytotoxic and possibly mutagenic effects that result from processes other than plasmid integration. In consequence, for any given enzyme used in REMI mutagenesis, the enzyme concentration that gives the highest number of transformants must be defined experimentally. Such optimal transformation conditions should give the highest probability of obtaining mutations caused by a single restriction enzyme-mediated integration of the selection marker.     

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.     

Restriction enzyme-mediated DNA integration in Coprinus cinereus

Restriction enzyme-mediated DNA integration (REMI) has recently received attention as a new technique for the generation of mutants by transformation in fungi. Here we analyse this method in the basidiomycete Coprinus cinereus using the homologous pab1 gene as a selectable marker and the restriction enzymes BamHI, EcoRI and PstI. Addition of restriction enzymes to transformation mixtures results in an earlier appearance of transformants and influences transformation rates in an enzyme- and concentration-dependent manner. Low concentrations of restriction enzyme result in increased numbers of transformants compared to no addition of enzymes. Transformation rates decrease with higher enzyme concentrations. If protoplasts are made from cells stored in the cold, the transformation rates drop drastically even in the presence of low amounts of enzyme. In several transformants, plasmid integration directly correlated with the action of restriction enzyme at random chromosomal restriction sites. In some cases, restriction enzymes appear to reduce the number of integration events per transformant. Simultaneously, mutation rates can be enhanced due to the presence of restriction enzymes. Although restriction enzymes clearly promote plasmid integration into the host genome they also have cytotoxic and possibly mutagenic effects that result from processes other than plasmid integration. In consequence, for any given enzyme used in REMI mutagenesis, the enzyme concentration that gives the highest number of transformants must be defined experimentally. Such optimal transformation conditions should give the highest probability of obtaining mutations caused by a single restriction enzyme-mediated integration of the selection marker. Received: 2 September 1996 / Accepted: 7 April 1997     

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura^? strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura⁺ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.     

Nonhomologous End Joining during Restriction Enzyme-Mediated DNA Integration in Saccharomyces cerevisiae

The BamHI restriction enzyme mediates integration of nonhomologous DNA into the Saccharomyces cerevisiae genome (R. H. Schiestl and T. D. Petes, Proc. Natl. Acad. Sci. USA 88:7585–7589, 1991). The present study investigates the mechanism of such events: in particular, the mediating activity of various restriction enzymes and the processing of resultant fragment ends. Our results show that in addition to BamHI, BglII and KpnI increase DNA integration efficiencies severalfold, while Asp718, HindIII, EcoRI, SalI, SmaI, HpaI, MscI, and SnaBI do not. Secondly, the three active enzymes stimulated integrations only of fragments containing 5′ or 3′ overhangs but not of blunt-ended fragments. Thirdly, integrations mediated by one enzyme and utilizing a substrate created by another required at least 2 bp of homology. Furthermore, an Asp718 fragment possessing a 5′ overhang integrated into a KpnI (isoschizomer) site possessing a 3′ overhang, most likely by filling of the 5′ overhang followed by 5′ exonuclease digestion to produce a 3′ end. We classified and analyzed the restriction enzyme-mediated integration events in the context of their genomic positions. The majority of events integrated into single sites. In the remaining 6 of 19 cases each end of the plasmid inserted into a different sequence, producing rearrangements such as duplications, deletions, and translocations.DNA double-strand breaks occur either as a result of assaults by external agents or spontaneously during DNA metabolism, repair, or replication. Double-strand breaks may cause genome rearrangements, such as deletions, duplications, and translocations, which have been implicated in carcinogenesis. For any cell, double-strand break repair is essential, since these cytotoxic DNA lesions may cause potentially lethal losses of chromosomes. In the yeast Saccharomyces cerevisiae, DNA repair enzymes encoded by genes belonging to the RAD52 epistasis group repair double-strand breaks by homologous recombination. This process requires homologous DNA sequences, usually present on sister chromatids and on homologous chromosomes in diploids. In mammalian cells, however, the majority of double-strand breaks are repaired by nonhomologous end joining (NHEJ) (32). This event in S. cerevisiae occurs either in rad52 mutants in the presence of homology (18) or in the wild type in the absence of homology (26, 36). Joining reactions of restriction enzyme-produced DNA ends have frequently been used to study NHEJ both in vivo and in vitro. NHEJ of substrates with defined terminal configurations produced by different enzyme digestions were studied in vitro in the presence of Xenopus laevis extracts (2, 30, 43) and in vivo in mammalian cells (32) and fission yeast cells (12). In S. cerevisiae, illegitimate repair of a double-strand break in a plasmid was studied by Mezard and Nicolas (25) and the repair of double-strand breaks produced by an inducible HO endonuclease in the absence of homology was studied by Moore and Haber (26) (for the conclusions of those studies see Discussion).Schiestl and Petes (36) studied illegitimate integration events by transforming a BamHI URA3 fragment into yeast cells lacking homology to the transforming DNA (in a ura3 deletion mutant), so that integration into the genome was by illegitimate recombination. With BamHI in the transformation mixture, the URA3 fragment integrated into genomic BamHI sites and the frequency of integration increased sixfold (36). These experiments suggest that the BamHI restriction enzyme can cut chromosomal DNA in vivo and thus mediate integration of the transforming DNA into that site. Subsequently, restriction enzyme-mediated integration (REMI) has been used in a variety of organisms for insertional mutagenesis. For example, Kuspa and Loomis (20) first adapted this technology to Dictyostelium discoideum, where previously cloning of developmental genes by complementation of mutant phenotypes was not feasible. Application of REMI has led to construction of REMI-restriction fragment length polymorphism maps (19, 23) and the cloning of most developmental genes in Dictyostelium. REMI has also been adapted successfully to the ascomycete Cochliobolus heterostrophus (24) and the maize pathogenic fungus Ustilago maydis (3) to tag genes by insertional mutagenesis.Here we find that enzymes vary in their ability to mediate integration into the yeast genome. Furthermore, we present model mechanisms based on the products created from various blunt 5′ protruding single strand (PSS) and 3′ PSS joining combinations.     

Phage lambda receptor chromosomes for DNA fragments made with restriction endonuclease I of Bacillus amyloliquefaciens H.

A lambda derivative with DNA resistant to attack by R. BamHI has been obtained following mutagenesis to remove a single target for this enzyme. The two central targets for R. BamHI were then introduced into the genome of this phage and the DNA between them removed in vitro to give a lambda insertion vector for fragments of DNA produced by digestion with R. BamHI.     

Mapping of BamHI and SmaI DNA restriction sites on the genome of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica

The DNA of the nuclear polyhedrosis virus of the alfalfa looper, Autographa californica (AcNPV), has been analyzed with restriction endonucleases BamHI and SmaI. The molecular weight of the BamHI fragments, SmaI fragments, and BamHI + SmaI fragments has been determined. The molecular weight of AcNPV DNA is calculated to be about 82 million. A presumptive physical map of the BamHI and SmaI restriction sites on the AcNPV genome has been constructed.     

Increased transformation frequency and tagging of developmental genes in Aspergillus nidulans by restriction enzyme-mediated integration (REMI)

We have used a plasmid containing the argB gene to transform an Aspergillus nidulansargB-deleted strain in the presence of restriction enzymes and show a 20- to 60-fold increase in transformation frequency via restriction enzyme-mediated integration (REMI). This procedure was used to try to tag new genes involved in the asexual development of this fungus. More than 2000 transformants isolated following electroporation of conidia and ～3700 transformants recovered following protoplast fusion were screened for sporulation defects. Unexpectedly, developmental mutants were obtained only when the protoplast fusion approach was used. Southern blot analysis of these mutants, and of randomly selected transformants obtained by electroporation, was consistent with the occurrence of single plasmid integration events in 33 and 65% of the cases, respectively. The argB marker was shown to be tightly linked to the mutant phenotype in only 62% of the mutants analyzed by sexual crosses. Partial DNA sequencing of a tagged gene, whose mutation delays asexual sporulation and results in a fluffy phenotype, showed no homology to previously reported sequences. Our results indicate that REMI can be used in A.?nidulans to increase the transformation frequency and illustrate the advantages and potential problems when using REMI to tag genes of interest in this and other fungi.     

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.     

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     

Restriction enzymes increase efficiencies of illegitimate DNA integration but decrease homologous integration in mammalian cells

Mammalian cells repair DNA double-strand breaks by illegitimate end-joining or by homologous recombination. We investigated the effects of restriction enzymes on illegitimate and homologous DNA integration in mammalian cells. A plasmid containing the neo^R expression cassette, which confers G418 resistance, was used to select for illegitimate integration events in CHO wild-type and xrcc5 mutant cells. Co-transfection with the restriction enzymes BamHI, BglII, EcoRI and KpnI increased the efficiency of linearized plasmid integration up to 5-fold in CHO cells. In contrast, the restriction enzymes did not increase the integration efficiency in xrcc5 mutant cells. Effects of restriction enzymes on illegitimate and homologous integration were also studied in mouse embryonic stem (ES) cells using a plasmid containing the neo^R gene flanked by exon 3 of Hprt. The enzymes BamHI, BglII and EcoRI increased the illegitimate integration efficiency of transforming DNA several-fold, similar to the results for CHO cells. However, all three enzymes decreased the absolute frequency of homologous integration ~2-fold, and the percentage of homologous integration decreased >10-fold. This suggests that random DNA breaks attract illegitimate recombination (IR) events that compete with homology search.     

Tn5 mutagenesis of the transforming genes of human adenovirus type 5

We have cloned the entire human adenovirus type 5 (Ad5) genome into the pBR322 plasmid in two segments: the BamHI-A fragment (21 kb) and the BamHI-B fragment (15 kb). We have also generated a series of clones with smaller Ad5 DNA inserts, all containing the left-end of the viral genome. One such clone, pXCl, containing the left 16% of the Ad5 DNA molecule, has been shown to transform rodent cells by DNA transfection. We have used the transposable element Tn5 as an insertion mutator to isolate pXCl mutants containing Tn 5 inserted at a large number of sites. By assaying transforming activity of selected pXC::Tn5 plasmids we have identified Ad5 sequences which are essential for DNA-mediated transformation. Our results with these mutants and with a plasmid pCDl, containing a deletion within the Ad5-transforming region, indicate that sequences present in both early region la and the N-terminal region of early region 1b are essential for DNA-mediated transformation.     

Dissecting the Molecular Origins of Specific Protein-Nucleic Acid Recognition: Hydrostatic Pressure and Molecular Dynamics

The fundamental processes by which proteins recognize and bind to nucleic acids are critical to understanding cellular function. To explore the factors involved in protein-DNA recognition, we used hydrostatic pressure to perturb the binding of the BamHI endonuclease to cognate DNA, both in experiment and in molecular dynamic simulations. A new technique of high-pressure gel mobility shift analysis was used to test the effects of elevated hydrostatic pressure on the binding of BamHI to its cognate recognition sequence. Upon application of a pressure of 500 bar, the equilibrium dissociation constant of BamHI binding to the cognate site was found to increase nearly 10-fold. A challenge has been to link this type of pure thermodynamic measurement to functional events occurring at the molecular level. Thus, we used molecular dynamic simulations at both ambient and elevated pressures to reveal details of the direct and water-mediated interactions between BamHI and cognate DNA, which allow explanation of the effects of pressure on site-specific protein-DNA binding and complex stability.     

Stable transformation and regulated expression of an inducible reporter construct inCandida albicans using restriction enzyme-mediated integration

To allow the regulated expression of cloned genes inCandida albicans, a plasmid was constructed using the inducible promoter of theC. albicans MAL2 gene. To demonstrate that theMAL2 promoter could regulate cloned genes placed under its control, a fusion construct was made with the coding sequence of theC. albicans URA3 gene. This plasmid was introduced into a Ura^– strain ofC. albicans using the process of restriction enzyme-mediated integration (REMI). This procedure involves the transformation of theBamHI-linearized plasmid in the presence ofBamHI enzyme. The majority of transformants generated contained insertions of the plasmid at chromosomalBamHI sites. All transformants examined were inducible forURA3 expression, which was determined by growth analysis and by measuring the level ofURA3 gene product activity. The Ura⁺ phenotype of the transformants was stable during growth under nonselective conditions. This system offers the advantages of stable transformation, easy recovery of integrated DNA, and inducible expression of genes inC. albicans.Deceased, December 15, 1995     

Characterization of eight excision plasmids of Pseudomonas syringae pv. phaseolicola

Summary Pseudomonas syringae pv. phaseolicola strain LR719 contains a 150 kilobase pair (kb) plasmid pMC7105, stably integrated into its chromosome. Occasionally, single colony isolates of this strain contain an excision plasmid. Eight unique excision plasmids were selected and characterized by BamHI restriction endonuclease and blot hybridization analyses. These plasmids ranged in size from 35 to 270 kb; the largest contained approximately 130 kb of chromosomal DNA sequences. Restriction maps of pMC7105 were developed to deduce the site of integration and to identify the fragments in which recombination occurred to produce each excision plasmid. The eight excision plasmids were arranged into five classes based on the sites where excision occurs. A 20 kb region of pMC7105, which includes BamHI fragment 9 and portions of adjacent fragments, is present in all excision plasmids and thought to contain the origin of replication. The site of integration on pMC7105 maps within BamHI fragment 8. This fragment shows homology with seven other BamHI fragments of pMC7105 and with five chromosomal fragments identified among the excision plasmids. The data strongly suggest that the integration of pMC7105 may have occurred at a repetitive sequence present on the chromosome and on the plasmid.     

Construction and BamHL analysis of chimeric plasmids containing EcoRL DNA fragments of the F sex factor.

The construction of seven chimeric plasmids (pRS series) carrying EcoRI endonuclease-generated segments of the F sex factor cloned onto the vector pSC101 is described. BamHI endonuclease analysis of these seven plasmids, the six previously described pRS plasmids (Skurray, R. A., Nagaishi, H., and Clark, A. J. (1976) Proc. Nat. Acad. Sci. USA73, 64–68) and F plasmid DNA has enabled a partial BamHI map of F to be constructed; the orientation of insertion of F DNA segments into the pSC101 vector was also established for nine of the pRS plasmids. Results indicate that in the absence of their normal promoter, F cistrons cloned into the EcoRI site of pSC101 are expressed regardless of orientation of insertion although there is a preferred orientation for high levels of expression.     

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.     

A helicase gene (helO) in Rhizobium meliloti WSM419

A 2.8 kb BamHI DNA fragment adjacent to a BamHI fragment containing actR-actS (a sensor/regulator pair required for low pH tolerance in Rhizobium meliloti WSM419) was cloned and sequenced. A computer predicted protein of 821 amino acids, designated HelO, showed extensive similarity with `DEAH' motif helicases. Expression of helO was higher at pH 7.0 than pH 5.8 and it did not require the product of the actR gene. Inactivation of helO by insertion of a Ω interposon at codon 40 did not affect nodulation, growth or tolerance to low pH, high temperature, osmolarity or elevated levels of copper or zinc.     

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.