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.     

Molecular cloning of chromosome I DNA from Saccharomyces cerevisiae: Isolation of the MAK16 gene and analysis of an adjacent gene essential for growth at low temperatures

MAK16 is an essential gene on chromosome I defined by the thermosensitive lethal mak16–1 mutation. MAK16 is also necessary for M double-stranded RNA replication at the permissive temperature for cell growth. As part of an effort to clone all the DNA from chromosome I, plasmids that complemented both the temperature-sensitive growth defect, and the M₁ replication defects of mak16–1 strains were isolated from a plasmid YCp50: Saccharomyces cerevisiae recombinant DNA library. The two plasmids analysed contained overlapping inserts that hybridized proportionally to strains carrying different dosages of chromosome I. Furthermore, integration of a fragment of one of these clones occurred at a site linked to ade1, confirming that this clone was derived from the appropriate region of chromosome I. An open reading frame adjacent to MAK16 potentially coding for a 468 amino acid protein was defined by sequence analysis. 185 amino acids of this open reading frame were replaced with a 1·2 kb fragment carrying the S. cerevisiae URA3 gene by a one-step gene disruption. The resulting strains grew at a rate indistinguishable from the wild type at 20°C, 30°C, or 37°C, but could not grow at 8°C. The deleted region is thus essential only at 8°C, and we name this gene LTE1 (low temperature essential).     

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.     

Salmon and Trout Analysis by PCR-RFLP for Identity Authentication

Raw and smoked Atlantic salmon (Salmo salar) and rainbow trout (Oncorhynchus mykiss) were differentiated by PCR amplification of a 950 bp conserved fragment of the mitochondrial 16S rRNA gene followed by restriction site analysis with the endonucleases Hpa I and Bst Ell. Salmon PCR products digested with Hpa I yielded two fragments of 804 bp and 146 bp, while trout PCR products were not cleaved by this enzyme. However, Bst Ell did not cleave salmon PCR products while two bands of 513 bp and 437 bp were produced when trout samples were cleaved with this enzyme. This genetic marker could be very useful for detecting fraudulent substitution of lower valued smoked trout.     

A series of yeast shuttle vectors for expression of cDNAs and other DNA sequences

Expression/shuttle vectors for the yeast Saccharomyces cerevisiae have usually been large plasmids with only one or a small number of sites that are suitable for cloning and expression. We report here the construction and properties of a series of 12 expression vectors with multiple (four to eight) unique sites in their polylinkers which allow directional cloning and expression of DNA sequences under four different promoters. Eleven of these plasmids replicate at high copy number in Escherichia coli, and all have the yeast TRP1 gene, and the 2 μm origin including REP3 sequence, allowing selection and high copy number replication in yeast. Six of the plasmids are designed for the construction and selection of cDNA libraries from various eukaryotic organisms, allowing directional cloning and expression of cDNAs. All of these six have similar polylinkers containing a unique promoter proximal EcoRI site and a unique promoter distal XhoI site, allowing for directional cloning and expression of ‘ZAP’-type cDNAs. cDNAs that complement a wide variety of yeast mutants can be selected from libraries constructed in this way. The four alternative promoters, ADH2, PGK, GAL10 and SV40 were compared for their relative activity, both in E. coli and in yeast. All yeast promoters showed substantial activity in E. coli with ADH2 showing the highest activity. ADH2 also was well-regulated in yeast, showing very high relative activity under derepressing conditions. cDNAs selected by genetic complementation from libraries constructed in these vectors should be easily subclonable into other vectors, allowing expression in different eukaryotic organisms, DNA sequencing or site-directed mutagenesis.     

EXPRESSION OF THE STA2 GLUCOAMYLASE GENE OF SACCHAROMYCES CEREVISIAE IN BREWERS' YEAST

Two fragments of DNA containing the Saccharomyces cerevisiae STA2 glucoamylase gene, with differing lengths of 5î non-coding DNA, were separately subcloned into a yeast centromeric plasmid. Of these two subclones, only the shorter one (containing 127 base-pairs of 5î non-coding DNA) was able to confer glucoamylase production on a standard laboratory strain of S. cerevisiae. The longer subclone (containing 465 bp of 5î non-coding DNA) did, however, confer glucoamylase production on a strain of S. cerevisiae lacking a functional STA10 gene (which encodes a repressor of STA2 gene expression). All-yeast plasmids lacking bacterial DNA were constructed from the two STA2 subclones for the transformation of a lager brewing yeast. Only the shorter STA2 subclone conferred glucoamylase activity on this yeast. The level of enzyme activity was comparable to that produced by the same yeast strain containing STA2 expressed from the PGK1 (that is, PGK1) promoter.     

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.     

PCR-RFLP analysis of the rpoB gene to distinguish the five species of Cronobacter

Members of the genus Cronobacter are opportunistic pathogens associated with life-threatening infections in immuno-compromised individuals. Polyphasic analysis has facilitated the classification of the novel genus Cronobacter containing five species. However, since this recent reclassification there are not many identification methods optimised for differentiation between the five Cronobacter species. This differentiation between the species is of importance as there are indications that the species may be diverse regarding their virulence. The aim of this study was to develop a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) protocol to differentiate between the five Cronobacter species. The rpoB gene of 49 Enterobacteriaceae strains, including 33 Cronobacter strains was amplified using conventional PCR, followed by digestion of these PCR products with restriction endonucleases MboI, HinP1I and Csp6I. The PCR-RFLP analysis with single digestions of each of the restriction endonucleases did not distinguish between all five Cronobacter species. This study describes the successful differentiation of the five Cronobacter species based on the amplification of the rpoB gene followed by the combined digestion with restriction endonucleases Csp6I and HinP1I. This PCR-RFLP assay is an accurate identification method that ensures rapid differentiation between the five species of Cronobacter.     

Cloning and characterization of a novel NAD+‐dependent glyceraldehyde‐3‐phosphate dehydrogenase gene from Candida glycerinogenes and use of its promoter

A 3950 bp genomic fragment from Candida glycerinogenes, WL2002‐5, containing the CgGAP gene encoding a glyceraldehyde‐3‐phosphate dehydrogenase homologous to GAP genes in other yeasts using degenerate primers, was cloned and characterized with inverse PCR. Sequence analysis revealed a 1164 bp open reading frame encoding a putative peptide of 387 deduced amino acids, with a molecular mass of 36 kDa. The CgGAP protein consisted of an N‐terminal NAD⁺‐binding domain and a central catalytic domain. Six stress‐response elements were found in the upstream region of the CgGAP gene. The influence of CgGAP on glycolysis was investigated. Functional analysis revealed that Saccharomyces cerevisiae transformed with CgGAP was restored to the wild‐type phenotype when cultured in high‐osmolarity medium, suggesting that it is a functional GAP protein. Promoter studies in S. cerevisiae using the green fluorescent protein (gfp) gene as a reporter showed that the GAP promoter (P_CgGAP) is constitutively expressed in S. cerevisiae cells grown on glucose. Copyright © 2013 John Wiley & Sons, Ltd.     

Functional analysis reports. Precise gene disruption in Saccharomyces cerevisiae by double fusion polymerase chain reaction

We adapted a fusion polymerase chain reaction (PCR) strategy to synthesize gene disruption alleles of any sequenced yeast gene of interest. The first step of the construction is to amplify sequences flanking the reading frame we want to disrupt and to amplify the selectable marker sequence. Then we fuse the upstream fragment to the marker sequence by fusion PCR, isolate this product and fuse it to the downstream sequence in a second fusion PCR reaction. The final PCR product can then be transformed directly into yeast. This method is rapid, relatively inexpensive, offers the freedom to choose from among a variety of selectable markers and allows one to construct precise disruptions of any sequenced open reading frame in Saccharomyces cerevisiae.     

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.     

Disruption of six novel ORFs on the left arm of chromosome XII reveals one gene essential for vegetative growth of Saccharomyces cerevisiae

Deletion via PCR‐mediated gene replacement, together with basic functional and bioinformatic analyses, have been performed on six novel open reading‐frames (ORFs) on the left arm of chromosome XII of Saccharomyces cerevisiae(YLL033w, YLL032c, YLL031c, YLL030c, YLL029w and YLL028w). ORF deletion was realized using either a short‐flanking homology (SFH) or a long‐flanking homology (LFH) replacement cassette in the diploid strain FY1679. Sporulation and tetrad analysis showed that YLL031c is the only essential gene of the six. Microscopic examination of the non‐growing spores carrying a disrupted copy of the essential gene showed that most of them were blocked after one or two cell divisions with heterogeneous bud size. The standard EUROFAN growth tests failed to reveal any obvious phenotype resulting from the deletion of each the five non‐essential ORFs. Bioinformatic analysis revealed that YLL029w is probably an aminopeptidase for mitochondrial or nuclear protein processing and YLL028w may be involved in drug resistance in S. cerevisiae. Replacement cassettes, comprising the promoter and terminator regions of each of the six ORFs, were cloned into pUG7 and demonstrated to efficiently mediate gene replacement in an alternative diploid strain, W303. All the cognate gene clones were constructed, using either PCR products amplified from genomic DNA, or gap‐repair. All clones and strains generated have been deposited in the EUROFAN genetic stock centre (EUROSCARF, Frankfurt). Copyright © 1999 John Wiley & Sons, Ltd.     

Cloning an Autonomous Replication Sequence from Aspergillus oryzae

DNA sequence from Aspergillus oryzae genome which, confers on pOK-1 the ability to replicate autonomously in Saccharomyces cerevisiae is described. A hybrid plasmid pOK-1 was constructed from pBR325 and YEp13 fragment containing Leu2⁺ gene. Asp. oryzae DNA and pOK-1 were cleaved with BamHI, ligated and introduced into Escherichia coli. About 600 recombinant plasmids were obtained. One of them transforms S. cerevisiae SHY3 to Leu2⁺ with middle efficiency. The plasmid designated as pSB1–2 contained two BamHI fragments (4.3 kb and 4.1 kb) of Asp. oryzae DNA.     

A new promoter-probe vector for Saccharomyces cerevisiae using fungal glucoamylase cDNA as the reporter gene

A system is described for the selection of DNA sequences showing promoter activity in the yeast Saccharomyces cerevisiae using a heterologous reporter enzyme which is efficiently secreted by the yeast host. A multicopy shuttle plasmid of the YEp-type was constructed so as to carry multiple unique cloning sites at the 5′ end of the Aspergillus awamori glucoamylase cDNA. Glucoamylase can only be expressed upon insertion at one of these unique cloning sites of a DNA fragment from any source, provided it is endowed with promoter function in S. cerevisiae. As the glucoamylase signal-peptide is functional in S. cerevisiae, the enzyme is efficiently secreted by the yeast transformants. This phenotype can be very easily detected on plate assays and accurately quantified by spectrophotometric analysis of the culture supernatant. Since S. cerevisiae naturally lacks amylolytic activity, any wild-type strain can be used as a host in this system. To evaluate the system, a DNA pool of random fusions was created by ligating sau 3A digested S. cerevisiae genomic DNA to the BglII-linearized vector. The resulting hybrid plasmids were transformed into S. cerevisiae and several transformants secreting glucoamylase to varying degrees were obtained.     

Sequence analysis of a 5·6 kb fragment of chromosome II from Saccharomyces cerevisiae reveals two new open reading frames next to CDC28

The sequence of a 5653 bp DNA fragment of the right arm of chromosome II of Saccharomyces cerevisiae contains two unknown open reading frames (YBR1212 and YBR1213) next to gene CDC28. Gene disruption reveals both putative genes as non-essential. ORF YBR1212 encodes a predicted protein with 71% similarity and 65% identity (total polypeptide of 376 aa) with the 378 aa Sur1 protein of S. cerevisiae, while the putative product of ORF YBR1213, which is strongly expressed, has 28% identity with a Lactococcus lactis-secreted 45 kDa protein and 24% identity with the Saccharomyces cerevisiae AGA1 gene product. The total sequence of the fragment has been submitted to the EMBL databank (accession number X80224).     

‘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.     

Expression Cassettes for Formaldehyde and Fluoroacetate Resistance,Two Dominant Markers in Saccharomyces cerevisiae

We employed two genes in constructing yeast expression cassettes for dominant, selectable markers. The Saccharomyces cerevisiae gene SFA1, encoding formaldehyde dehydrogenase, was placed under the control of the GPD1 promoter and CYC1 terminator. The Moraxella sp. strain B gene dehH1, encoding fluoroacetate dehalogenase, was placed under the control of both the GPD1 and CYC1 promoters. With these constructs it was possible to select directly for yeast strains resistant to either formaldehyde or fluoroacetate. Both selective agents are completely metabolized and inexpensive, making them very useful in the pursuit of yeast gene functions and for industrial applications. An additional advantage of the formaldehyde dehydrogenase marker is that it is an S. cerevisiae gene, thus allowing ‘all yeast’ constructs. © 1997 John Wiley & Sons, Ltd.     

Development of a Polymerase Chain Reaction System for the Detection of Dog and Cat Meat in Meat Mixtures and Animal Feed

The identification of species origin of meat represents a considerable problem for food and animal feed analysis. In the present study a PCR‐mediated method for the detection of dog and cat meat was developed. For this the cytochrome b gene sequence of both species was analyzed by restriction fragment length polymorphism (RFLP) analysis. The use of the restriction enzymes Alu I and Hae III yielded specific restriction profiles characteristic for each species. The meat of both species could additionally be differentiated with species‐specific oligonucleotide primers based on specific parts of the cytochrome b gene sequences characteristic for dog and cat. The use of these oligonucleotide primers allowed a direct identification of dog and cat meat in meat mixtures even after heat treatment.