A New Efficient Gene Disruption Cassette for Repeated Use in Budding Yeast (original) (raw)

Abstract

The dominant _kan_r marker gene plays an important role in gene disruption experiments in budding yeast, as this marker can be used in a variety of yeast strains lacking the conventional yeast markers. We have developed a loxP-kanMX-loxP gene disruption cassette, which combines the advantages of the heterologous _kan_r marker with those from the Cre- lox P recombination system. This disruption cassette integrates with high efficiency via homologous integration at the correct genomic locus (routinely 70%). Upon expression of the Cre recombinase the kanMX module is excised by an efficient recombination between the loxP sites, leaving behind a single loxP site at the chromosomal locus. This system allows repeated use of the _kan_r marker gene and will be of great advantage for the functional analysis of gene families.

Introduction

The yeast Saccharomyces cerevisiae will be the first eukaryotic organism for which the entire sequence of the genome will be determined ( 1 ). This will result in the identification of all 6500–7000 genes from this organism. The next big challenge is the functional characterization of the unknown gene products. The first step towards a functional analysis of a protein is the complete deletion of the corresponding gene on the chromosome (null mutant) using the one-step gene transplacement method ( 2 ). Classically, DNA fragments flanking the gene of interest are cloned left and right of a yeast marker gene and, after transformation of this construct into yeast, homologous recombination between the flanking regions results in a deletion of the gene and the simultaneous integration of the marker gene. Because yeast has very efficient mechanisms for homologous recombination, it has been possible to reduce the length of the flanking DNA regions to 30–45 bp, allowing the construction of gene disruption cassettes by the polymerase chain reaction (PCR) ( 2 ; see also Fig 2 ). This system allows construction of gene disruption cassettes without cloning steps and only requires the DNA sequence of the relevant chromosomal locus. The system could be improved significantly by using a heterologous marker gene. This avoids the problem of gene conversion associated with the use of yeast marker genes and recipient yeast strains not completely deleted for the marker gene. The _kan_r gene from the Escherichia coli transposon Tn903 when expressed in yeast renders the transformants resistant to the aminoglycoside antibiotic G418 ( 6 , 7 ). Very recently the _kan_r gene was fused to the TEF promoter and terminator sequences from the filamentous fungus Ashbya gossypii yielding the kanMX expression module. PCR-mediated generation of disruption cassettes carrying this completely heterologous expression module allows efficient gene disruptions ( 8 ).

An important result from the yeast sequence analysis is the finding that a substantial portion of genes are duplicated in the genome ( 1 ). For example, in silico analysis of the available sequence data revealed the existence of at least 15 proteins belonging to the HXT family of hexose transporters ( 9 ). A second example are the flocculation genes, which constitute a new subtelomeric gene family ( 10 ). Thus, for the functional analysis of an unknown gene all members of a gene family might have to be disrupted before the real phenotype of a null mutant can be studied. For example, only if all seven hexose transporter genes, HXT1-HXT7 , are deleted can reduced growth on low and high glucose media be observed ( 11 ).

As the number of marker genes is limited, efficient procedures for marker rescue will be very important for functional analysis projects. For marker rescue, in the presently available disruption cassettes the marker genes are surrounded by direct repeats of 40–1100 bp. After gene disruption homologous recombination between the two repeats results in marker removal, leaving behind a single repeat at the deleted gene locus ( 8 , 12 , 13 ). The disadvantage of the low mitotic recombination frequency of ∼10 −4 has been overcome in the case of the URA3 marker gene by selecting for the loss of the marker on 5-fluoroorotic acid (5-FOA) ( 12 , 13 ). Alternatively the Cre- loxP recombination system of bacteriophage P1 has been shown to mediate efficient recombination between loxP sites flanking a marker gene in yeast, resulting in excision of the marker gene ( 14 ).

We have constructed the gene disruption cassette loxP-kanMX-loxP , which combines the advantages of the heterologous _kan_r marker with those of the Cre- loxP system. The PCR-generated gene disruption cassette integrates with high efficiency via homologous recombination at the correct genomic locus (routinely 70%). In addition, the fast marker rescue procedure allows repeated use of the _kan_r marker.

Materials and Methods

Strains and media

Strain CEN.PK2 (MATa/MATα leu2–3,112/leu2–3,112 ura3–52 /ura3–52 trp1–289/trp1–289 his3-Δ1/ his3-Δ1 MAL2–8 c SUC2) was obtained from Karl-Dieter Entian (University of Frankfurt, Germany). The E.coli strain XL1-Blue was from Stratagene (Heidelberg, Germany). Yeast and E.coli media were prepared as described ( 15 ). For selection for G418 resistance after yeast transformations the YPD plates were supplemented with 200 mg/l Geneticin ® (G-418 sulphate from Gibco BRL, Germany; the activity of this chemical may be batch dependent). Selection for the _kan_r gene in E.coli was on YT plates supplemented with 50 mg/l kanamycin sulphate (Fluka, Germany) ( 8 ).

Plasmid construction

The loxP-kanMX-loxP module containing plasmid pUG6 was constructed by modification of plasmid pFA6-kanMX4 ( 8 ). Four oligonucleotides (319, 320, 321 and 322) were made, which after annealing formed the two oligonucleotide duplexes 319/320 and 321/322, each carrying the 34 bp loxP sequence and appropriate restriction enzyme sites at either end ( Table 2 ). The oligonucleotide duplex 319/320 was ligated into the Sal I/ Bgl II-cleaved pFA6-kanMX4 plasmid. Next, this modified vector was cleaved with Sac I and Eco RV and the oligonucleotide duplex 321/322 was inserted, generating plasmid pUG6 ( Fig. 1 A). The modified regions were sequenced using the SP6 and T7 primers to verify correct insertion of the oligonucleotide duplexes.

The cre recombinase expression vector pSH47 (6.78 kb) was obtained by placing a 1.2 kb Sal I- Xho I DNA fragment comprising the cre open reading frame (ORF) from plasmid pBS39 (obtained from Brasch; 16 ) into a Sal I-cleaved p416 /GAL1 plasmid ( 17 ).

PCR-mediated generation of the _loxP-kan_r-loxP gene disruption cassette

We pooled two preparative PCR reactions for a gene disruption. A 100 µl preparative PCR reaction contained 10 µl 10× GoldStar PCR buffer [750 mM Tris-HCl, pH 9.0, 200 mM ammonium sulphate, 0.1% (w/v) Tween 20], 200 µM dNTP mix (200 µM each of dATP, dCTP, dGTP and dTTP), 30–60 ng pUG6 template DNA, 100 pmol each primer, 1.5 mM MgCl 2 and 0.25 U GoldStar polymerase (Eurogentec, Belgium). The PCR conditions were: 98°C for 1.0 min, 50°C for 1.0 min, 72°C for 2.5 min (40 cycles). Aliquots of 5 µl of the reaction were quantified on an agarose gel to verify the amount of DNA. The two PCR reactions were pooled and the DNA was extracted with phenol/chloroform (1:1) and ethanol precipitated. The pellet was resuspended in 13 µl water and 0.5 µl were analyzed on an agarose gel. Typically, the concentration of the PCR product was 0.3–0.5 µg/µl. About 0.5 µl DNA were used for cloning the disruption cassette into pUG7 (Güldener and Hegemann, unpublished results), while 12 µl (∼5 µg) were used for yeast transformation.

Yeast transformation

The yeast transformation procedure used was a slightly modified version of the protocol described by Gietz and colleagues ( 18 ). Cells from an overnight culture were resuspended in 50 ml YPD (start OD 600 0.2) and grown to an OD 600 of 0.7–1.0. The cells were harvested by centrifugation (Heraeus Minifuge RF; 4000 r.p.m., 5 min) and resuspended in 10 ml sterile distilled water. The cells were harvested by centrifugation, resuspended in 1 ml water and transferred to a 1.5 ml Eppendorf tube. The cells were harvested by centrifugation (Heraeus Biofuge 13; 5000 r.p.m., 1 min) and resuspended in 1.5 ml freshly prepared sterile TE/LiOAc (prepared from 10× concentrated stocks; 10 × TE = 0.1 M Tris-HCl, 0.01 M EDTA, pH 7.5; 10× LiOAc = 1 M LiOAc adjusted to pH 7.5 with dilute acetic acid). The cells were harvested again and resuspended in 200 µl TE/LiOAc (cell concentration should be ∼2 × 10 9 cells/ml). For a gene disruption experiment ∼5 µg DNA (12 µl) of the disruption cassette were mixed with 50 µg (5µl) of freshly denatured salmon sperm DNA (10 mg/ml, boiled for 20 min in a water bath, then chilled in ice/water; the stock solution was prepared as described by Schiestl and Gietz; 19 ) and 50 µl cells in TE/LiOAc were added and mixed with caution (no vortexing!). Immediately 300 µl of freshly prepared sterile 40% PEG 4000 (prepared from stock solutions: 50% PEG 4000, 10× TE, 10× LiOAc, 8:1:1 v/v, pH 7.5) were added and carefully mixed (no vortexing!). Cells were incubated for 30 min at 30°C with constant agitation. Cells were incubated for 15 min at 42°C, then 800 µl sterile water were added, mixed and cells were collected by centrifugation (Biofuge 13; 13 000 r.p.m., 10 s). Cells were resuspended in 1 ml YPD (no vortexing!) and incubated for 2–3 h at 30°C. Cells were collected by centrifugation, resuspended in 200 µl YPD and plated onto YPD plus G418 plates (200 µg/ml G-418; Gibco BRL). Plates were incubated at 30 °C until colonies appeared. In cases where the background growth was too strong after 24–48 h, the microcolonies were replica-plated onto new YPD plus G418 plates. After re-streaking transformants onto YPD plus G418 plates only transformants growing well were chosen for further analysis.

Figure 1

( A ) Map of plasmid pUG6 carrying the loxP-kanMX-loxP disruption module. The oligonucleotide duplex 319/320 was cloned into the Sal I /Bgl II sites of pFA6-kanMX4 ( 8 ), while the oligonucleotide duplex 321/322 was inserted into the Xho I /Eco RV sites of this vector. The two loxP sites are flanking as direct repeats the _kan_r marker gene ( _kan_r ) plus TEF2 promoter ( T .-P.) and TEF2 terminator ( T .-T.). Unique restriction enzymes are indicated in bold. ( B ) Gene disruption using the loxP-kanMX-loxP disruption cassette. For a gene disruption experiment two oligonucleotides were used that carry at their 3′-end a segment (arrow) homologous to sequences left and right of the loxP-kanMX-loxP module on plasmid pUG6 and at their 5′-end a segment (shaded box) homologous to the ORF to be disrupted (for oligonucleotides see Table 1 ). Plasmid pUG6 was used as PCR template to generate the disruption cassette.

Figure 2

_kan_r marker rescue by expression of the Cre recombinase. The diploid kan + yeast strain with the relevant genotype ORF/ORF:: loxP-kanMX-loxP was transformed with plasmid pSH47. Transformants were grown on glucose plates and then shifted to galactose medium to induce expression of the Cre recombinase. The Cre-induced recombination process between the two loxP sites removes the marker gene resulting in the genotype ORF/ORF ::loxP .

Transformation of yeast strain CEN.PK2 with the loxP-kanMX-loxP disruption cassette typically yields 10–100 kan + transformants/µg DNA, while transformation of a control plasmid (pRS316, a CEN, ARSH4, URA3 plasmid) routinely gives 2–10 × 10 4 transformants/µg DNA.

Detection of gene targeting events and of Cre-mediated marker rescue by PCR and Southern analysis

Detection of the correct gene disruption of ORF N2809 was done by either diagnostic PCR or Southern analysis. For PCR yeast cells were taken directly from a YPD+G-418 plate. The PCR primers were a ‘start’ primer and a ‘stop’ primer, located 400–500 nt upstream of the ATG and of the stop codon respectively of the disrupted gene, and two primers located in the 5′ (kanRE) and in the 3′ regions (kanFW) of the _kan_r gene respectively ( Table 1 ). Genomic integration of the disruption cassette was verified using the ORF ‘start’ and the kanRE primers as well as with the ORF ‘stop’ and the kanFW primers. After the _kan_r gene was excised by the Cre recombinase the ORF ‘start’ and ‘stop’ primers were used. The PCR reaction was as described for the generation of the disruption cassette. The conditions were: 94°C for 2.0 min (hot start); then 40 cycles of 94°C for 1.5 min, 45°C for 2.0 min and 72°C for 2 min. About 15 µl of the PCR reaction were loaded on an agarose gel.

Analysis of the disruption events of ORF N3265 was by Southern analysis using a PCR-generated probe (0.46 kb) located 5′ of the ORF. The PCR was carried out as described above using primers 400 and 424 ( Table 1 ). The probe was labeled with digoxigenin and the Southern analysis was performed according to the supplier's instructions (Boehringer, Mannheim, Germany).

Results and Discussion

Gene disruption using the loxP-kanMX-loxP disruption cassette

The aim of this work was to combine the great advantages of the dominant _kan_r marker system for gene disruption and the Cre -loxP recombination system for marker rescue. To this end the plasmid pFA6-kanMX4 ( 8 ) was modified by integrating two 34 bp loxP sequences as direct repeats left and right of the kanMX module using appropriate oligonucleotide duplexes ( Fig. 1 A). The new vector, pUG6, has all the restriction sites present in pFA6-kanMX4 excluding those used for integration of the loxP sequences ( Fig. 1 A). Thus pUG6 can be used in the classical way for gene disruption experiments. For this, homologous DNA fragments are cloned left and right of the loxP-kanMX-loxP module, followed by a second restriction enzyme digest to generate the gene disruption cassette. Alternatively, short homologous sequences needed for homologous recombination can be fused to the loxP-kanMX-loxP module via a PCR reaction ( 3 , 4 ). Routinely we use two oligonucleotides of ∼60 nt in length, comprised of a 20 nt long segment homologous to sequences left or right of the kanMX module at their 3′-ends and of a 40 nt long segment homologous to sequences left or right of the gene to be deleted at their 5′-ends ( Fig. 1 B). The oligonucleotides also hybridize to the pFA6-kanMX vectors and thus can be used to generate disruption cassettes without the loxP sites if required.

To evaluate the efficiency of the loxP-kanMX-loxP module in gene disruption experiments the pUG6 vector was used as target for the PCR-mediated generation of disruption cassettes for three different ORFs. The ORFs N0868, N0901 and N3216 had been identified during systematic sequencing work on chromosome XIV ( 20 ; Sen-Gupta et al. , unpublished results; Düsterhöft, unpublished results). The corresponding oligonucleotides for generation of the disruption cassettes were 59–62 nt in length ( Table 1 ). Transformation into the diploid yeast strain CEN.PK2 yielded sufficient numbers of kan + transformants (48–370 transformants; Table 2 ). Meanwhile, another 10 gene disruptions have been performed using the loxP-kanMX-loxP module, yielding on average 10–100 kan + tranformants/µg disruption cassette (data not shown). Diagnostic PCR and Southern analysis of a selected number of transformants verified the correct integration of the cassette into the chromosomal target in ∼70% of the kan + clones on average ( Table 2 ). The data show that the percentage of correctly integrated disruption cassettes is independent of the number of transformants obtained in a particular experiment, suggesting that the transformation efficiency is not affected by the integration efficiency at the correct locus. The results of these disruption experiments demonstrate the power of the loxP-kanMX-loxP module in gene disruption experiments.

Oligonucleotides used in this study (the loxP sequences are underlined)

Table 1

Oligonucleotides used in this study (the loxP sequences are underlined)

The _kan_r marker gene can be efficiently rescued

To use the _kan_r marker repeatedly for several gene disruptions in one strain it is necessary to eliminate the marker from the successfully disrupted gene. The heterozygous disruption strain CEN.HE18, in which one of the two copies of ORF N2809 was disrupted by the loxP-kanMX-loxP cassette ( N2809/N2809::loxP-kanMX-loxP ), was transformed with the cre expression plasmid pSH47, which carries the URA3 marker gene and the cre gene under the control of the inducible GAL1 promoter. Expression of the Cre recombinase was induced by shifting cells from YPD (glucose) to YPG (galactose) medium ( Fig 2 ). Growth for only 2 h in galactose medium after transfer from glucose medium was sufficient to remove the _kan_r marker gene in ∼80–90% of the cells, as detected by plating cells on YPD and replica-plating the colonies onto YpD plus G418. Correct loss of the _kan_r marker gene was verified by diagnostic PCR and Southern analysis, which confirmed that in all kan − clones tested the _kan_r marker gene had been excised, leaving behind a single loxP site at the chromosomal N2809 locus (data not shown, but see Fig. 3 ). The cre expression plasmid was removed from this strain by streaking cells on plates containing 5-fluoroorotic acid to counterselect for the loss of the plasmid, yielding strain CEN.HE18-1 (relevant genotype N2809/-N2809::loxP ).

Yeast transformation and integration efficiencies

Table 2

Yeast transformation and integration efficiencies

Figure 3

Verification of marker rescue by diagnostic PCR and Southern analysis. Yeast strain CEN.HE18-2, heterozygous on chromosome XIV for N2809 (N2809/N2809::loxP) and N3265 (N3265IN3265::loxP-kanMX-loxP) , was transformed with the cre expression plasmid pSH47 and transformants were shifted to galactose medium to induce cre expression. Twenty-four strains which were kan − were analyzed for the structure of the disrupted ORFs N3265 and N2809 (analysis of six strains is shown here). ( A ) Two verification primers (366 and 367) were used in a PCR to verify N2809::loxP . All clones analyzed showed a band of 0.86 kb as expected for an intact N2809::loxP locus, while the band of 1.56 kb indicative of the undisruptedN2809 allele was barely visible. ( B ) Southern analysis of the N3265 locus revealed in all six clones tested the presence of the non-disrupted N3265 allele (fragment size 3.48 kb) and the disrupted N3265::loxP allele (fragment size 2.16 kb).

A second gene can be disrupted in a loxP − carrying yeast strain

The central question was, whether it would be possible to create a second gene disruption in strain CEN.HEl8-1 using the loxP-kanMX-loxP cassette and subsequently remove the _kan_r marker again by induction of the Cre recombinase. To test this, strain CEN.HE18-1 was transformed with a PCR-generated disruption cassette with homology to 3′- and 5′-ends of ORF N3265 . This ORF is also located on chromosome XIV and in opposite orientation ∼93 kb away from the already disrupted ORF N2809 . From 68 kan + transformants obtained, two of four clones tested by diagnostic PCR were found to be correctly integrated into the genome (data not shown). Two ORF N3265 -disrupted transformants (strain CEN.HE18-2) were transformed with the cre expression plasmid pSH47 to induce recombination and subsequent loss of the _kan_r marker to generate N3265::loxP . The two plasmid-transformed strains were pre-grown on selective glucose plates to select for presence of the plasmid and then shifted to liquid YPD (glucose) and grown overnight. The cells were then shifted to liquid YPG (galactose) for a 30, 60, 90 or 120 min period to follow the kinetics of Cre induction. This would indicate whether a longer incubation with the Cre recombinase would be toxic to the cells due to recombination between the loxP sites at the N3265 locus and the N2809 locus leading to loss of all the genetic material located between both loci. Afterwards cells were re-shifted to YPD plates and analyzed for the presence or absence of the _kan_r marker. Incubation in YPD showed that ∼11% of cells tested had lost the _kan_r marker, indicating that the galactose-regulated GAL1 promoter is not completely shut off in glucose medium and thus a small amount of Cre recombinase is produced in YPD medium. Thirty minutes after the shift to YPG ∼80% of the cells were kan − and this number did not change significantly at later time points. Southern analysis of the kan − strains revealed that all 72 kan − clones tested (from time points 0 and 30 min, 24 clones each; from time points 60 and 90 min, 12 clones each) had lost the _kan_r gene, yielding a 2.16 kb band ( Fig. 3 ) indicative of the expected N3265::loxP disruption. As the N2809::loxP locus could be a second potential target for a recombination event we analyzed this locus by diagnostic PCR. Twenty-four kan − clones analyzed showed a band of 0.86 kb in length, indicating that this locus was intact and that no recombination had occurred between this loxP site and the other site at N3265 ( Fig. 3 ).

These results show that all Cre-induced recombination events analyzed involved only the two loxP sites flanking the kanMX module. The single loxP site located ∼93 kb away from the second integration site is obviously too far away to be relevant for the Cre-induced recombination process. Further studies are required to determine precisely how close the loxP-kanMX-loxP cassette can be placed to a single loxP site without affecting the desired recombination process. Furthermore, it needs to be determined whether location of the loxP-kanMX-loxP cassette and the single loxP site on the same chromosome (see below), on homologous chromosomes or on non-homologous chromosomes is relevant for the outcome of the recombination process. In mouse embryonic stem cells the frequency of a Cre-mediated translocation between loxP sites located on non-homologous chromosomes was determined to be 1 in ∼1200–2400 cells expressing Cre recombinase ( 21 ).

The _kan_r marker is the recommended marker for the European Project for the Functional Analysis of Unknown Genes (EURO-FAN) (Guidelines for the EUROFAN B0 program: ORF Deletants, Plasmid Tools, Basic Functional Analysis by A. Wach, A. Brachat and P. Philippsen, personal communication). The results presented here show that the _kan_r marker, which is very efficient in gene disruption experiments, can be successfully combined with the Cre -loxP recombination system to generate the reusable disruption cassette loxP-kanMX-loxP . Repeated use of the _kan_r marker is now possible and will be of great advantage for the analysis of gene families. After a first gene disruption, tetrad analysis can be used to determine whether the disrupted gene is essential or not. In cases where it is known that the gene to be disrupted is non-essential, gene disruption and Cre-mediated recovery of the _kan_r marker can be performed directly in haploid yeast strains. ORFs N2809 and N3265 are both non-essential genes and were also deleted in a two-step process using the loxP-kanMX-loxP cassette and the Cre recombinase in a haploid CEN.PK-derived strain. No reduction in viability was observed after Cre-mediated removal of the _kan_r gene of the second deletion, indicating also that no recombination event had taken place between the single loxP site at the N2809 locus and the two loxP sites at N3265 (data not shown).

Further improvements to this system are possible. For example, the high efficiency of the disruption cassettes should allow simultaneous transformation of two disruption cassettes for two different genes. It has been shown previously that co-transformation efficiency is 30–40% in yeast ( 22 ). Such a ‘co-gene disruption’ would be even more efficient when the two disruption cassettes contain different markers. Very recently a new heterologous HIS marker has been constructed which might be suitable for this purpose (Wach, Brachat, Alberti-Segui and Philippsen, personal communication). After Cre activation both markers can be recovered and used again.

In summary, the loxP-kanMX-loxP cassette in conjunction with the Cre recombinase will significantly facilitate multiple gene disruptions. It is particularly useful in yeast strains carrying incomplete deletions of the normally used homologous marker genes, as well as in industrially used yeast strains, which often lack the standard auxotrophic markers present in laboratory strains.

Acknowledgements

We thank Dr Ursula Fleig for critical reading of the manuscript. We thank Dr Brasch for plasmid PBS39 and Drs Wach and Philippsen for the pFA plasmid series. This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 272, T.P. A1) and of the BMBF (no. FKZ 0310577) to JHH.

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