Additional modules for versatile and economical PCR-based gene deletion and modification in Saccharomyces cerevisiae

An important recent advance in the functional analysis of Saccharomyces cerevisiae genes is the development of the one-step PCR-mediated technique for deletion and modification of chromosomal genes. This method allows very rapid gene manipulations without requiring plasmid clones of the gene of interest. We describe here a new set of plasmids that serve as templates for the PCR synthesis of fragments that allow a variety of gene modifications. Using as selectable marker the S. cerevisiae TRP1 gene or modules containing the heterologous Schizosaccharomyces pombe his5⁺ or Escherichia coli kan^r gene, these plasmids allow gene deletion, gene overexpression (using the regulatable GAL1 promoter), C- or N-terminal protein tagging [with GFP(S65T), GST, or the 3HA or 13Myc epitope], and partial N- or C-terminal deletions (with or without concomitant protein tagging). Because of the modular nature of the plasmids, they allow efficient and economical use of a small number of PCR primers for a wide variety of gene manipulations. Thus, these plasmids should further facilitate the rapid analysis of gene function in S. cerevisiae. © 1998 John Wiley & Sons, Ltd.     

Disruption of Six Novel Yeast Genes Reveals Three Genes Essential for Vegetative Growth and One Required for Growth at Low Temperature

We describe here the construction of six deletion mutants and their basic phenotypic analysis. Six open reading frames (ORFs) from chromosome X, YJR039w, YJR041c, YJR043c, YJR046w, YJR053w and YJR065c, were disrupted by deletion cassettes with long (LFH) or short (SFH) flanking regions homologous to the target locus. The LFH deletion cassette was made by introducing into the kanMX4 marker module two polymerase chain reaction (PCR) fragments several hundred base pairs (bp) in size homologous to the promoter and terminator regions of a given ORF. The SFH gene disruption construct was obtained by PCR amplification of the kanMX4 marker with primers providing homology to the target gene. The region of homology to mediate homologous recombination was about 70 bp. Sporulation and tetrad analysis revealed that ORFs YJR041c, YJR046w and YJR065c are essential genes. Complementation tests by corresponding cognate gene clones confirmed this observation. The non-growing haploid segregants were observed under the microscope. The yjr041cΔ haploid cells gave rise to microcolonies comprising about 20 to 50 cells. Most yjr046wΔ cells were blocked after one or two cell cycles with heterogeneous bud sizes. The yjr065cΔ cells displayed an unbudded spore or were arrested before completion of the first cell division cycle with a bud of variable size. The deduced protein of ORF YJR065c, that we named Act4, belongs to the Arp3 family of actin-related proteins. Three other ORFs, YJR039w, YJR043c and YJR053w are non-essential genes. The yjr043cΔ cells hardly grew at 15°C, indicating that this gene is required for growth at low temperature. Complementation tests confirmed that the disruption of YJR043c is responsible for this growth defect. In addition, the mating efficiency of yjr043cΔ and yjr053wΔ cells appear to be moderately a ffected. © 1997 John Wiley & Sons, Ltd.     

PCR-synthesis of marker cassettes with long flanking homology regions for gene disruptions in S. cerevisiae

A PCR-method for fast production of disruption cassettes is introduced, that allows the addition of long flanking homology regions of several hundred base pairs (LFH-PCR) to a marker module. Such a disruption cassette was made by linking two PCR fragments produced from genomic DNA to kanMX6, a modification of dominant resistance marker making S. cerevisiae resistant to geneticin (G418). In a first step, two several hundred base pairs long DNA fragments from the 5′- and 3′-region of a S. cerevisiae gene were amplified in such a way that 26 base pairs extensions homologous to the kanMX6 marker were added to one of their end. In a second step, one strand of each of these molecules then served as a long primer in a PCR using kanMX6 as template. When such a LFH-PCR-generated disruption cassette was used instead of a PCR-made disruption cassette flanked by short homology regions, transformation efficiencies were increased by at least a factor of thirty. This modification will therefore also help to apply PCR-mediated gene manipulations to strains with decreased transformability and/or unpredictable sequence deviations.     

Discovery of genes involved in mitosis,cell division,cell wall integrity and chromosome segregation through construction of Schizosaccharomyces pombe deletion strains

The fission yeast model system Schizosaccharomyces pombe is used to study fundamental biological processes. To continue to fill gaps in the Sz. pombe gene deletion collection, we constructed a set of 90 haploid gene deletion strains covering many previously uncharacterized genes. To begin to understand the function of these genes, we exposed this collection of strains to a battery of stress conditions. Using this information in combination with microscopy, proteomics and mini‐chromosome loss assays, we identified genes involved in cell wall integrity, cytokinesis, chromosome segregation and DNA metabolism. This subset of non‐essential gene deletions will add to the toolkits available for the study of biological processes in Sz. pombe. Copyright © 2016 John Wiley & Sons, Ltd.     

New constructs and strategies for efficient PCR-based gene manipulations in yeast

Gene disruption and tagging can be achieved by homologous recombination in the yeast genome. Several PCR-based methods have been described towards this end. However these strategies are often limited in their applications and/or their efficiencies and may be technically demanding. Here we describe two plasmids for C-terminal tagging of proteins with the IgG binding domain of the Staphyloccocus aureus protein A. We also present simple and reliable strategies based on PCR to promote efficient integration of exogenous DNA into the yeast genome. These simple methods are not limited to specific strains or markers and can be used for any application requiring homologous recombination such as gene disruption and epitope tagging. These strategies can be used for consecutive introduction of various constructs into a single yeast strain. © 1998 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.     

A versatile toolbox for PCR-based tagging of yeast genes: new fluorescent proteins, more markers and promoter substitution cassettes

Tagging of genes by chromosomal integration of PCR amplified cassettes is a widely used and fast method to label proteins in vivo in the yeast Saccharomyces cerevisiae. This strategy directs the amplified tags to the desired chromosomal loci due to flanking homologous sequences provided by the PCR-primers, thus enabling the selective introduction of any sequence at any place of a gene, e.g. for the generation of C-terminal tagged genes or for the exchange of the promoter and N-terminal tagging of a gene. To make this method most powerful we constructed a series of 76 novel cassettes, containing a broad variety of C-terminal epitope tags as well as nine different promoter substitutions in combination with N-terminal tags. Furthermore, new selection markers have been introduced. The tags include the so far brightest and most yeast-optimized version of the red fluorescent protein, called RedStar2, as well as all other commonly used fluorescent proteins and tags used for the detection and purification of proteins and protein complexes. Using the provided cassettes for N- and C-terminal gene tagging or for deletion of any given gene, a set of only four primers is required, which makes this method very cost-effective and reproducible. This new toolbox should help to speed up the analysis of gene function in yeast, on the level of single genes, as well as in systematic approaches.     

Quantitative analysis of yeast gene function using competition experiments in continuous culture

One possible route to the evaluation of gene function is a quantitative approach based on the concepts of metabolic control analysis (MCA). An important first step in such an analysis is to determine the effect of deleting individual genes on the growth rate (or fitness) of S. cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, we employed competition experiments in chemostat culture to measure the proportion of deletion mutants relative to that of a standard strain by using a quantitative PCR method. In this paper, we show that both densitometry and GeneScan^™ analysis can be used with similar accuracy and reproducibility to determine the proportions of (at least) two strains simultaneously, in the range 10–90% of the total cell population. Furthermore, we report on a model competition experiment between two diploid nuclear petite mutants, homozygous for deletions in the cox5a or pet191 genes, and the standard strain (ho::kanMX4/ho::kanMX4) in chemostat cultures under six different physiological conditions. The results indicate that competition experiments in continuous culture are a suitable method to distinguish quantitatively between deletion mutants that qualitatively exhibit the same phenotype. © 1998 John Wiley & Sons, Ltd.     

Suitability of replacement markers for functional analysis studies inSaccharomyces cerevisiae

The complete yeast sequence contains a large proportion of genes whose biological function is completely unknown. One approach to elucidating the function of these novel genes is by quantitative methods that exploit the concepts of metabolic control analysis. An important first step in such an analysis is to determine the effects of deleting individual genes on the growth rate (or fitness) of Saccharomyces cerevisiae. Since the specific growth-rate effects of most genes are likely to be small, they are most readily determined by competition against a standard strain in chemostat cultures where the true steady state demanded by metabolic control analysis may be achieved. We have constructed two different standard strains in which the HO gene is replaced by either HIS3 or kanMX. We demonstrate that HO is a selectively neutral site for gene replacement. However, there is a significant marker effect associated with HIS3 which, moreover, is dependent on the physiological conditions used for the competition experiments. In contrast, the kanMX marker exhibited only a small effect on specific growth rate (≤±4%). These data suggest that nutritional markers should not be used to generate deletion mutants for the quantitative analysis of gene function in yeast but that kanMX replacements may be used, with confidence, for such studies. © 1997 John Wiley & Sons, Ltd.     

Functional analysis of yeast essential genes using a promoter-substitution cassette and the tetracycline-regulatable dual expression system

A promoter-substitution cassette has been constructed that allows one-step substitution of chromosomal gene promoters for the tetracycline-regulatable tetO promoter in yeast cells, which uses kanMX4 as selective marker for geneticin resistance. Oligonucleotides for PCR amplification of the cassette are designed to allow homologous recombination through short flanking regions of homology with the upstream sequences of the chromosomal gene, upon transformation of target cells. By testing three essential genes of chromosome XV (YOL135c, YOL142w and YOL144w), the system causes tetracycline-dependent conditional growth of the cells, being modulatable by intermediate concentrations of the effector. Analysis of terminal phenotypes of the promoter-substituted cells in the presence of the antibiotic may facilitate functional analysis of essential orphan genes. © 1998 John Wiley & Sons, Ltd.     

Properties and Heterologous Expression of the Glucose Transporter GHT1 from Schizosaccharomyces pombe

Genomic DNA of the Schizosaccharomyces pombe glucose transporter, GHT1, was obtained by complementation of the glucose transport deficient Sz. pombe strain YGS-5. Here we describe the GHT1 gene that encodes a protein of 565 amino acids with a corresponding molecular mass of 62·5 kDa. This eukaryotic glucose transporter contains 12 putative transmembrane segments and is homologous to the HXT multigene family of S. cerevisiae with several amino acid motifs of this sugar transporter family. It is also homologous to other sugar carriers from human, mouse and Escherichia coli. The function of the Ght1 protein as a glucose transporter was proved both by homologous and heterologous expression in the Sz. pombe mutant YGS-5 and in the S. cerevisiae hxt mutant RE700A, respectively. Both transformed yeast strains transported d -glucose with substrate specificity similar to that in Sz. pombe wild-type cells. Moreover, the cells of the two transformed yeast strains accumulated 2-deoxy-d -glucose, a non-metabolizable d -glucose analogue, with an efficiency similar to Sz. pombe wild-type cells. The ability of the S. cerevisiae mutant RE700A to accumulate 2DG in an Δμdependent manner after transformation with GHT1 provides evidence that the Sz. pombe transporter catalyses an energy-dependent uptake of glucose. The sequence of GHT1 was deposited at EMBL, Outstation EBI, Accession Number X91218. ©1997 John Wiley & Sons, Ltd.     

Designer deletion and prototrophic strains derived from Saccharomyces cerevisiae strain W303‐1a

We report the construction of Saccharomyces cerevisiae strains isogenic to W303‐1a that are designed to allow efficient genetic analysis. To facilitate the generation of null alleles of target genes by PCR‐mediated gene disruption, we constructed designer deletion alleles of the ARG4, TRP1 and URA3 genes. In addition, a single pair of oligonucleotide primers were designed that can be used to amplify any of several marker genes for use in PCR‐mediated gene disruption. A new version of the ‘reusable’ hisG‐URA3‐hisG cassette was constructed for use in PCR‐mediated gene disruption. Finally, to facilitate the formation of isogenic diploids by selection, we constructed strains that contain combinations of wild‐type alleles of ADE2, HIS3, LEU2, TRP1 and URA3. Copyright © 1999 John Wiley & Sons, Ltd.     

Plasmids with E2 epitope tags: tagging modules for N‐ and C‐terminal PCR‐based gene targeting in both budding and fission yeast,and inducible expression vectors for fission yeast

A single‐step PCR‐based epitope tagging enables fast and efficient gene targeting with various epitope tags. This report presents a series of plasmids for the E2 epitope tagging of proteins in Saccharomyces cerevisiae and Schizosaccharomyces pombe. E2Tags are 10‐amino acids (epitope E2a: SSTSSDFRDR)‐ and 12 amino acids (epitope E2b: GVSSTSSDFRDR)‐long peptides derived from the E2 protein of bovine papillomavirus type 1. The modules for C‐terminal tagging with E2a and E2b epitopes were constructed by the modification of the pYM‐series plasmid. The N‐terminal E2a and E2b tagging modules were based on pOM‐series plasmid. The pOM‐series plasmids were selected for this study because of their use of the Cre–loxP recombination system. The latter enables a marker cassette to be removed after integration into the loci of interest and, thereafter, the tagged protein is expressed under its endogenous promoter. Specifically for fission yeast, high copy pREP plasmids containing the E2a epitope tag as an N‐terminal or C‐terminal tag were constructed. The properties of E2a and E2b epitopes and the sensitivity of two anti‐E2 monoclonal antibodies (5E11 and 3F12) were tested using several S. cerevisiae and Sz. pombe E2‐tagged strains. Copyright © 2009 John Wiley & Sons, Ltd.     

N‐ICE plasmids for generating N‐terminal 3 × FLAG tagged genes that allow inducible,constitutive or endogenous expression in Saccharomyces cerevisiae

PCR‐mediated homologous recombination is a powerful approach to introduce epitope tags into the chromosomal loci at the N‐terminus or the C‐terminus of targeted genes. Although strategies of C‐terminal epitope tagging of target genes at their loci are simple and widely used in yeast, C‐terminal epitope tagging is not practical for all proteins. For example, a C‐terminal tag may affect protein function or a protein may get cleaved or processed, resulting in the loss of the epitope tag. Therefore, N‐terminal epitope tagging may be necessary to resolve these problems. In some cases, an epitope tagging strategy is used to introduce a heterologous promoter with the epitope tag at the N‐terminus of a gene of interest. The potential issue with this strategy is that the tagged gene is not expressed at the endogenous level. Another strategy after integration is to excise the selection marker, using the Cre‐LoxP system, leaving the epitope tagged gene expressed from the endogenous promoter. However, N‐terminal epitope tagging of essential genes using this strategy requires a diploid strain followed by tetrad dissection. Here we present 14 new plasmids for N‐terminal tagging, which combines two previous strategies for epitope tagging in a haploid strain. These ‘N‐ICE’ plasmids were constructed so that non‐essential and essential genes can be N‐terminally 3 × FLAG tagged and expressed from an inducible promoter (GAL1), constitutive promoters (CYC1 or PYK1) or the endogenous promoter. We have validated the N‐ICE plasmid system by N‐terminal tagging two non‐essential genes (SET1 and SET2) and two essential genes (ERG11 and PKC1). Copyright © 2016 John Wiley & Sons, Ltd.     

Divergence and conservation of SUP2(SUP35) gene of yeasts Pichia pinus and Saccharomyces cerevisiae

SUP2(SUP35) is an omnipotent suppressor gene, coding for an EF-1α-like protein factor, intimately involved in the control of translational accuracy in yeast Saccharomyces cerevisiae. In the present study a SUP2 gene analogue from yeast Pichia pinus was isolated by complementation of the temperature-sensitive sup2 mutation of S. cerevisiae. The nucleotide sequence of the SUP2 gene of P. pinus codes for a protein of 82·4 kDa, exceeding the Sup2 protein of S. cerevisiae by 6 kDa. Like the SUP2 gene product of S. cerevisiae, the Sup2 protein of P. pinus represents a fusion of a unique N-terminal part of a region homologous to EF-1α. The comparison of amino acid sequences of the Sup2 proteins reveals high conservations (76%) of the C-terminal region and low conservation (36%) of the N-terminal part where, in addition, the homologous correspondence is ambiguous. Proteins related to the Sup2 of S. cerevisiae where found in P. pinus and some other yeast species by the immunoblotting technique. The relation between the evolutionary conservation of different regions of the Sup2 protein and their functional significance is discussed.     

Efficient gene targeting in Kluyveromyces lactis

Integration of a DNA fragment in a host genome requires the action of a double-strand break (DSB) repair mechanism. Homologous recombination (HR) is initiated by binding of Rad52p to DNA ends and results in targeted integration. Binding of the Ku heterodimer (Ku70p/Ku80p) results in random integration via non-homologous end joining (NHEJ). In contrast to Saccharomyces cerevisiae, the budding yeast Kluyveromyces lactis shows variable, but in general low, gene targeting efficiency. To study and to improve gene targeting efficiency, K. lactis has been used as a model. The KlRAD51, KlRAD52 and KlKU80 genes have been isolated and deletion mutants for these genes have been constructed. Efficiency of gene targeting was determined at the KlADE2 locus using targeting constructs with different lengths of homologous flanking sequences. In wild-type K. lactis, the gene targeting efficiency ranged from 0% with 50 to 88% with 600 bp flanks. The Klku80 mutant, however, showed >97% gene targeting efficiency independently of the size of the homologous flanks. These results demonstrate that deletion of the NHEJ mechanism results in a higher gene targeting efficiency. Furthermore, increased gene targeting efficiency was achieved by the transformation of wild-type K. lactis with the KlADE2 deletion construct in the presence of excess small DNA fragments. Using this method, PCR-generated deletion constructs containing only 50 bp of homologous flanking sequences resulted in efficient targeted gene replacement.