‘Marker Swap’ Plasmids: Convenient Tools for Budding Yeast Molecular Genetics

One-step gene disruption constructs for disruption of HIS3, LEU2, TRP1 or URA3 with each of the other three markers have been constructed. All of these constructs have been tested and found to effectively convert markers either in gene disruptions or on plasmids. The ‘swapped’ strains allow the unambiguous genetic analysis of synthetic phenotypes with multiple genes, even if the original gene disruptions were made with the same marker. They also allow introduction of multiple plasmids in a single transformant, even if the original plasmids had the same marker, and allow transformation of plasmids into strains containing gene disruptions made with the same marker that is on the plasmids. These ‘marker-swap’ plasmids therefore eliminate the need for much subcloning to change markers. Marker-swapped alleles are acceptably stable mitotically and meiotically for most applications.© 1997 John Wiley & Sons, Ltd.     

A family of vectors that facilitate transposon and insertional mutagenesis of cloned genes in yeast

This report describes two sets of plasmid vectors that facilitate the identification of regions of complementation in cloned genomic inserts via transposon or insertional mutagenesis. The first set contains ARS-H4 CEN6, a yeast selectable nutritional marker (HIS3, TRP1 or URA3), and neo for selection in Escherichia coli. These plasmids lack the Tn3 transposition immunity region present in pBR322 derived vectors, and are permissive recipients for Tn3 transposon mutagenesis. The second family of plasmids described facilitate gene disruption procedures performed in vitro. These vectors carry disruption cassettes that contain different yeast selectable markers (HIS3, LEU2, TRP1 or URA3) adjacent to the Tn5 neo gene. These genes can be excised as a cassette on a common restriction fragment and introduced into any desired restriction site with selection for kanamycin resistance.     

Targeted gene replacement at the URA3 locus of the basidiomycetous yeast Pseudozyma antarctica and its transformation using lithium acetate treatment

The basidiomycetous yeast Pseudozyma antarctica is a remarkable producer of industrially valuable enzymes and extracellular glycolipids. In this study, we developed a method for targeted gene replacement in P. antarctica. In addition, transformation conditions were optimized using lithium acetate, single‐stranded carrier DNA and polyethylene glycol (lithium acetate treatment), generally used for ascomycetous yeast transformation. In the rice‐derived P. antarctica strain GB‐4(0), PaURA3, a homologue of the Saccharomyces cerevisiae orotidine‐5′‐phosphate decarboxylase gene (URA3), was selected as the target locus. A disruption cassette was constructed by linking the nouseothricine resistance gene (natMX4) to homologous DNA fragments of PaURA3, then electroporated into the strain GB‐4(0). We obtained strain PGB015 as one of the PaURA3 disruptants (Paura3Δ::natMX4). Then the PCR‐amplified PaURA3 fragment was introduced into PGB015, and growth of transformant colonies but not background colonies was observed on selective media lacking uracil. The complementation of uracil‐auxotrophy in PGB015 by introduction of PaURA3 was also performed using lithium acetate treatment, which resulted in a transformation efficiency of 985 CFU/6.8 μg DNA and a gene‐targeting ratio of two among 30 transformants. Copyright © 2017 John Wiley & Sons, Ltd.     

Analysis of protein function in clinical C. albicans isolates

Clinical isolates are prototrophic and hence are not amenable to genetic manipulation using nutritional markers. Here we describe a new set of plasmids carrying the NAT1 (nourseothricin) drug resistance marker (Shen et al., 2005 ), which can be used both in clinical isolates and in laboratory strains. We constructed novel plasmids containing HA–NAT1 or MYC–NAT1 cassettes to facilitate PCR‐mediated construction of strains with C‐terminal epitope‐tagged proteins and a NAT1–pMet3–GFP plasmid to enable conditional expression of proteins with or without the green fluorescent protein fused at the N‐terminus. Furthermore, for proteins that require both the endogenous N‐ and C‐termini for function, we have constructed a GF–NAT1–FP cassette carrying truncated alleles that facilitate insertion of an intact, single copy of GFP internal to the coding sequence. In addition, GFP–NAT1, RFP–NAT1 and M‐Cherry–NAT1 plasmids were constructed, expressing two differently labelled gene products for the study of protein co‐expression and co‐localization in vivo. Together, these vectors provide a useful set of genetic tools for studying diverse aspects of gene function in both clinical and laboratory strains of C. albicans. Copyright © 2012 John Wiley & Sons, Ltd.     

pMPY-ZAP: A reusable polymerase chain reaction-directed gene disruption cassette for Saccharomyces cerevisiae

Gene disruption is an important method for genetic analysis in Saccharomyces cerevisiae. We have designed a polymerase chain reaction-directed gene disruption cassette that allows rapid disruption of genes in S. cerevisiae without previously cloning them. In addition, this cassette allows recycling of URA3, generating gene disruptions without the permanent loss of the ura3 marker. An indefinite number of disruptions can therefore be made in the same strain.     

Identification of auxotrophic mutants of the yeast Kluyveromyces marxianus by non‐homologous end joining‐mediated integrative transformation with genes from Saccharomyces cerevisiae

The isolation and application of auxotrophic mutants for gene manipulations, such as genetic transformation, mating selection and tetrad analysis, form the basis of yeast genetics. For the development of these genetic methods in the thermotolerant fermentative yeast Kluyveromyces marxianus, we isolated a series of auxotrophic mutants with defects in amino acid or nucleic acid metabolism. To identify the mutated genes, linear DNA fragments of nutrient biosynthetic pathway genes were amplified from Saccharomyces cerevisiae chromosomal DNA and used to directly transform the K. marxianus auxotrophic mutants by random integration into chromosomes through non‐homologous end joining (NHEJ). The appearance of transformant colonies indicated that the specific S. cerevisiae gene complemented the K. marxianus mutant. Using this interspecific complementation approach with linear PCR‐amplified DNA, we identified auxotrophic mutations of ADE2, ADE5,7, ADE6, HIS2, HIS3, HIS4, HIS5, HIS6, HIS7, LYS1, LYS2, LYS4, LYS9, LEU1, LEU2, MET2, MET6, MET17, TRP3, TRP4 and TRP5 without the labour‐intensive requirement of plasmid construction. Mating, sporulation and tetrad analysis techniques for K. marxianus were also established. With the identified auxotrophic mutant strains and S. cerevisiae genes as selective markers, NHEJ‐mediated integrative transformation with PCR‐amplified DNA is an attractive system for facilitating genetic analyses in the yeast K. marxianus. Copyright © 2013 John Wiley & Sons, Ltd.     

Yeast Saccharomyces cerevisiae selectable markers in pUC18 polylinkers

A set of plasmids was constructed that contain the yeast selectable markers HIS3, LEU2, TRP1 or URA3 embedded in the multiple cloning site of pUC18.     

Designed construction of recombinant DNA at the ura3Δ0 locus in the yeast Saccharomyces cerevisiae

Recombinant DNAs are traditionally constructed using Escherichia coli plasmids. In the yeast Saccharomyces cerevisiae, chromosomal gene targeting is a common technique, implying that the yeast homologous recombination system could be applied for recombinant DNA construction. In an attempt to use a S. cerevisiae chromosome for recombinant DNA construction, we selected the single ura3Δ0 locus as a gene targeting site. By selecting this single locus, repeated recombination using the surrounding URA3 sequences can be performed. The recombination system described here has several advantages over the conventional plasmid system, as it provides a method to confirm the selection of correct recombinants because transformation of the same locus replaces the pre‐existing selection marker, resulting in the loss of the marker in successful recombinations. In addition, the constructed strains can serve as both PCR templates and hosts for preparing subsequent recombinant strains. Using this method, several yeast strains that contained selection markers, promoters, terminators and target genes at the ura3Δ0 locus were successfully generated. The system described here can potentially be applied for the construction of any recombinant DNA without the requirement for manipulations in E. coli. Interestingly, we unexpectedly found that several G/C‐rich sequences used for fusion PCR lowered gene expression when located adjacent to the start codon. Copyright © 2013 John Wiley & Sons, Ltd.     

IpO: plasmids and methods for simplified,PCR‐based DNA transplant in yeast

Many yeast experiments require strains modified by recombinant DNA methods. Some experiments require precise insertion of a DNA segment into the genome without a selectable marker remaining. For these applications, we developed a new PCR‐based method for marker‐free DNA transplant. The current PCR‐based method requires the labour‐intensive construction of a PCR template plasmid with repeats of the DNA segment flanking URA3. The design of a new vector, IpO, reduces the work in cloning a single copy of the DNA segment between overlapping URA3 fragments present in the vector. Two PCRs are performed that capture the DNA segment and one or the other URA3 fragment. When the PCR products are co‐transformed into yeast, recombination between the overlapping URA3 fragments restores URA3 and transposes the cloned DNA segment inside out, creating a repeat‐URA3‐repeat cassette. Sequences designed into the PCR primers target integration of the cassette into the genome. Subsequent selection with 5‐fluoro‐orotic acid yields strains that have 'popped out' URA3 via recombination between the DNA repeats, with the result being the precise insertion of the DNA segment minus the selectable marker. An additional advantage of the IpO method is that it eliminates PCR artifacts that can plague the current method's repeat‐containing templates. Copyright © 2014 John Wiley & Sons, Ltd.     

New tools for phenotypic analysis in Candida albicans: the WAR1 gene confers resistance to sorbate

Availability of the complete sequence of the Candida albicans genome allows for global gene analysis. We designed a gene deletion method to facilitate such studies. First, we constructed C. albicans strains that are both Deltaura3 and Deltatrp1. Second, we designed a system that relies on in vitro recombination, using the Gateway((R)) technology, for efficient generation of deletion cassettes. They are generated in two steps: (a) upstream and downstream DNA fragments of the chromosomal region to be deleted are amplified by PCR and introduced into two separate entry vectors; (b) the second step involves a quadruple recombination event including the two entry vectors, a plasmid bearing a marker of interest and a destination vector, in order to generate a plasmid containing the deletion cassette. The deletion plasmid contains very rare restriction sites for convenient excision of the knockout cassette. Selection in C. albicans can be performed with one of the following markers: the C. albicans URA3 gene, a modified S. cerevisiae TRP1 gene or the mycophenolic acid resistance (MPA(R)) gene. Upon integration into the genome, these markers can be removed by the use of 5-fluoroorotic acid (URA3), 5-fluoroanthranilic acid (TRP1) or the FLP recombinase (MPA(R)). Using this approach, we show that removal of the C. albicans orf19.1035 gene results in sensitivity to the weak acid sorbate, while its overexpression increases resistance to this compound. We named it WAR1, in analogy to its S. cerevisiae orthologue.     

Transformation of Candida albicans with a synthetic hygromycin B resistance gene

Synthetic genes that confer resistance to the antibiotic nourseothricin in the pathogenic fungus Candida albicans are available, but genes conferring resistance to other antibiotics are not. We found that multiple C. albicans strains were inhibited by hygromycin B, so we designed a 1026 bp gene (CaHygB) that encodes Escherichia coli hygromycin B phosphotransferase with C. albicans codons. CaHygB conferred hygromycin B resistance in C. albicans transformed with ars2‐containing plasmids or single‐copy integrating vectors. Since CaHygB did not confer nourseothricin resistance and since the nourseothricin resistance marker SAT‐1 did not confer hygromycin B resistance, we reasoned that these two markers could be used for homologous gene disruptions in wild‐type C. albicans. We used PCR to fuse CaHygB or SAT‐1 to approximately 1 kb of 5′ and 3′ noncoding DNA from C. albicans ARG4, HIS1 and LEU2, and introduced the resulting amplicons into six wild‐type C. albicans strains. Homologous targeting frequencies were approximately 50–70%, and disruption of ARG4, HIS1 and LEU2 alleles was verified by the respective transformants' inabilities to grow without arginine, histidine and leucine. CaHygB should be a useful tool for genetic manipulation of different C. albicans strains, including clinical isolates. Copyright © 2010 John Wiley & Sons, Ltd.     

Small epitope‐linker modules for PCR‐based C‐terminal tagging in Saccharomyces cerevisiae

PCR‐mediated gene modification is a powerful approach to the functional analysis of genes in Saccharomyces cerevisiae. One application of this method is epitope‐tagging of a gene to analyse the corresponding protein by immunological methods. However, the number of epitope tags available in a convenient format is still low, and interference with protein function by the epitope, particularly if it is large, is not uncommon. To address these limitations and broaden the utility of the method, we constructed a set of convenient template plasmids designed for PCR‐based C‐terminal tagging with 10 different, relatively short peptide sequences that are recognized by commercially available monoclonal antibodies. The encoded tags are FLAG, 3 × FLAG, T7, His‐tag, Strep‐tag II, S‐tag, Myc, HSV, VSV‐G and V5. The same pair of primers can be used to construct tagged alleles of a gene of interest with any of the 10 tags. In addition, a six‐glycine linker sequence is inserted upstream of these tags to minimize the influence of the tag on the target protein and maximize its accessibility for antibody binding. Three marker genes, HIS3MX6, kanMX6 and hphMX4, are available for each epitope. We demonstrate the utility of the new tags for both immunoblotting and one‐step affinity purification of the regulatory particle of the 26S proteasome. The set of plasmids has been deposited in the non‐profit plasmid repository Addgene ( http://www.addgene.org ). Copyright © 2009 John Wiley & Sons, Ltd.     

Construction of a URA3 deletion strain from the allotetraploid bottom-fermenting yeast Saccharomyces pastorianus

The bottom‐fermenting lager yeast Saccharomyces pastorianus has been proposed to be allotetraploid, containing two S. cerevisiae (Sc)‐type and two S. bayanus (Sb)‐type chromosomes. This chromosomal constitution likely explains why recessive mutants of S. pastorianus have not previously been reported. Here we describe the construction of a ura3 deletion strain derived from the lager strain Weihenstephan34/70 by targeted transformation and subsequent loss of heterozygosity (LOH). Initially, deletion constructs of the Sc and Sb types of URA3 were constructed in laboratory yeast strains in which a TDH3p‐hygro allele conferring hygromycin B resistance replaced ScURA3 and a KanMX cassette conferring G‐418 resistance replaced SbURA3. The lager strain was then transformed with these constructs to yield a heterozygous URA3 disruptant (ScURA3⁺/Scura3Δ::TDH3p‐hygro, SbURA3⁺/Sbura3Δ::KanMX), which was plated on 5‐fluoroorotic acid (5‐FOA) plates to generate the desired Ura^– homozygous disruptant (Scura3Δ::TDH3p‐hygro/Scura3Δ::TDH3p‐hygro Sbura3Δ::KanMX/Sbura3Δ::KanMX) through LOH. This ura3 deletion strain was then used to construct a bottom‐fermenting yeast transformant overexpressing ATF1 that encodes an enzyme that produces acetate esters. The ATF1‐overexpressing transformant produced significantly more acetate esters than the parent strain. The constructed ura3? lager strain will be a useful host for constructing strains of relevance to brewing. Copyright © 2012 John Wiley & Sons, Ltd.     

Isolation and characterization of Candida kefyr orotidine‐5′‐phosphate decarboxylase (URA3) gene

Candida kefyr is a common yeast species that can be found in fermented milk and cheeses. As a first step to developing a gene transfer system for C. kefyr, the orotidine‐5′‐phosphate decarboxylase (URA3) gene was cloned, using degenerate PCR and genome walking. The uninterrupted open reading frame of the C. kefyr URA3 gene spans 801 bp, corresponding to 267 amino acid residues. The functionality of the gene was confirmed by complementation of ura3 auxotrophs of C. albicans and Saccharomyces cerevisiae. Phylogenetic analysis of the deduced amino acid sequence indicated that it shares a high degree of homology with other Candida URA3 homologues. The GenBank Accession No. of the C. kefyr URA3 gene is FJ914763. Copyright © 2009 John Wiley & Sons, Ltd.     

Characterization of different promoters for designing a new expression vector in Saccharomyces cerevisiae

The widely used pESC vector series (Stratagene, La Jolla, CA, USA) with the bidirectional GAL1/GAL10 promoter provides the possibility of simultaneously expressing two different genes from a single vector in Saccharomyces cerevisiae. This system can be induced by galactose and is repressed by glucose. Since S. cerevisiae prefers glucose as a carbon source, and since its growth rate is higher in glucose than in galactose‐containing media, we compared and evaluated seven different promoters expressed during growth on glucose (pTEF1, pADH1, pTPI1, pHXT7, pTDH3, pPGK1 and pPYK1) with two strong galactose‐induced promoters (pGAL1 and pGAL10), using lacZ as a reporter gene and measuring LacZ activity in batch and continuous cultivation. TEF1 and PGK1 promoters showed the most constant activity pattern at different glucose concentrations. Based on these results, we designed and constructed two new expression vectors which contain the two constitutive promoters, TEF1 and PGK1, in opposite orientation to each other. These new vectors retain all the features from the pESC–URA plasmid except that gene expression is mediated by constitutive promoters. Copyright © 2010 John Wiley & Sons, Ltd.     

Multipurpose vectors designed for the fast generation of N- or C-terminal epitope-tagged proteins

In this paper are described a set of new high-copy-number yeast vectors, which are specially designed for the conditional expression of epitope-tagged proteins in vivo. One of the major advantages of these plasmids is that they allow polymerase chain reaction-amplified open reading frames to be automatically fused in frame with the epitope-coding sequence, avoiding longer procedures such as site-directed mutagenesis. This heterologous construction can be realized either at the 5′-end of the coding sequence, in the pYeF1 vector, or at its 3′-end, in pYeF2, generating N- or C-terminal tagged proteins, respectively. Moreover, to increase the usefulness of the method, derivatives of the two basic URA3-borne pYeF1 and pYeF2 were constructed, carrying either the HIS3 or TRP1 gene as a marker of selection. These vectors could be of use for the purpose of functional analysis of the newly discovered genes resulting from the systematic sequencing of the yeast genome. Here, we present results showing the functional expression and the efficient immunoprecipitation of the epitope-tagged Rna15 protein, which is involved in Saccharomyces cerevisiae mRNA stability.     

A recyclable Candida albicans URA3 cassette for PCR product-directed gene disruptions

For some time, gene disruptions in Candida albicans have been made with the hisG-URA3-hisG ('Ura-blaster') cassette, which can be re-used in successive transformations of a single strain after homologous excision of URA3. However, the hisG repeats are too large for efficient PCR amplification of the entire cassette, so it cannot be used for PCR product-directed gene disruptions. We describe here a gene disruption cassette, URA3-dpl200, with 200 bp flanking repeats that permit efficient PCR amplification. After transformation and integration to produce both arg5::URA3-dpl200 and rim101::URA3-dpl200 alleles, we find that arg5::dpl200 and rim101::dpl200 segregants, respectively, can be obtained. We have used the cassette to create rim101::dpl200/rim101::URA3-dpl200 mutants exclusively through PCR product-directed disruption.     

Use of polymerase chain reaction epitope tagging for protein tagging in Saccharomyces cerevisiae

Epitope tagging is the insertion of a short stretch of amino acids constituting an epitope into another protein. Tagged proteins can be identified by Western, immunoprecipitation and immunofluorescence assays using pre-existing antibodies. We have designed vectors containing the URA3 gene flanked by direct repeats of epitope tags. We use the polymerase chain reaction (PCR) to amplify the tag-URA3-tag cassette such that the ends of the PCR fragments possess homology to the gene of interest. In vivo recombination is then used to direct integration of the fragment to the location of interest, and transformants are selected by their Ura⁺ phenotype. Finally, selection for Ura^? cells on 5-fluoro-orotic acid plates yields cells where recombination between the repeated epitopes has ‘popped out’ the URA3 gene, leaving a single copy of the epitope at the desired location. PCR epitope tagging (PET) provides a rapid and direct technique for tagging that does not require any cloning steps. We have used PET to tag three Saccharomyces cerevisiae proteins, Cln1, Sic1 and Est1.     

A novel kanamycin/G418 resistance marker for direct selection of transformants in Escherichia coli and different yeast species

We have developed a set of cloning vectors possessing a modified Tn903 kanamycin resistance gene that enables the selection of both kanamycin‐resistant transformants in Escherichia coli and G418‐resistant transformants in the yeasts Saccharomyces cerevisiae, Hansenula polymorpha and Pichia pastoris. Expression of this gene in yeast is controlled by the H. polymorpha glyceraldehyde‐3‐phosphate dehydrogenase promoter, while expression in E. coli is governed by an upstream E. coli lacZ promoter. Applicability of the vectors for gene disruption in H. polymorpha and S. cerevisiae was demonstrated by inactivation of the HpMAL1 and URA3 genes, respectively. One of the vectors possesses a H. polymorpha ARS allowing plasmid maintenance in an episomal state. The small size of the vectors (2–2.5 kb) makes them convenient for routine DNA cloning. In addition, we report a novel approach for construction of gene disruption cassettes. Copyright © 2009 John Wiley & Sons, Ltd.