Você está na página 1de 5

SHORT TECHNICAL REPORTS

System of centromeric, episomal, and The plasmids and sequence files


can be obtained by contacting the
integrative vectors based on drug resistance authors (e-mails: taxis@embl.de or
knop@embl.de) or by sending a letter
markers for Saccharomyces cerevisiae with a self-addressed envelope to Dr.
Michael Knop, EMBL Cell Biology
Christof Taxis and Michael Knop and Biophysics Unit, Meyerhofstr.
EMBL, Heidelberg, Germany 1, D-69117 Heidelberg, Germany.
Standard mail is preferred.
BioTechniques 40:73-78 (January 2006)
doi 10.2144/000112040
MATERIALS AND METHODS
Integrative, centromeric, and episomal plasmids are essential for easy, fast, and reliable
genetic manipulation of yeast. We constructed a system of shuttle vectors based on the Construction of Plasmids
widely used plasmids of the pRS series. We used genes conferring resistance to Geneticin
(kanMX4), nourseothricin (natNT2), and hygromycin B (hphNT1) as markers. The Standard techniques were used
centromeric and episomal plasmids that we constructed can be used the same way as the tra- for DNA manipulations (16). The
ditional auxotrophic marker-based shuttle vectors (pRS41x and pRS42x series). Additionally, integrative plasmids are based on
we created a set of nine yeast integrative vectors with the three dominant markers. These pRS303, pRS305, or pRS306 (1). First,
plasmids allow for direct integration in the LEU2, URA3, and HIS3 locus of any yeast strain the multicloning sites (MCS) of these
and the concomitant partial deletion of the gene. This prevents multiple integrations and plasmids were destroyed, followed by
allows for the rapid identification of correct integrants. The set of new vectors consider- insertion of the drug resistance marker
ably enhances the flexibility of genetic manipulations and gene expression in yeast. Most in the HIS3, LEU2, or URA3 gene. The
notably, the new vectors allow one to work with natural yeast isolates, which do not contain centromeric and episomal plasmids
auxotrophic markers. were derived from plasmids pRS416
and pRS426, respectively (1,2). The
URA3 gene and the flanking promoter
and terminator regions in these
plasmids were replaced by the three
INTRODUCTION possible. To circumvent these problems,
different dominant selection markers.
we constructed a complete set of
A detailed description of the plasmid
The yeast Saccharomyces cerevisiae integrative, centromeric, and episomal
constructions is provided as supple-
is a widely used experimental system plasmids based on the pRS series of
mentary material, which is available
for basic research in cell biology. plasmids containing the three different
online at www.BioTechniques.com.
Numerous researchers use it because of dominant drug resistance markers
the ease of genetic manipulations. Two Geneticin (kanMX4), nourseothricin
different techniques are often used for (natNT2), and hygromycin B (hphNT1). Yeast Strains and Growth
genetic manipulations. One is the use of The centromeric and episomal plasmids Conditions
vectors such as the integrative, centro- can be used similarly to conventional Yeast strain ESM356 (17) was used
meric, and episomal plasmids of the plasmids. YPD (yeast extract, peptone, for in-yeast ligation by homologous
pRS series (1,2) for gene cloning and dextrose) medium supplemented with recombination. YPD and synthetic drop-
the second is the use of drug resistance the appropriate drug is used for plasmid out media were prepared as previously
markers for the deletion or tagging of selection, instead of synthetic medium described (18). The following drug
genes (313). The latter technique has lacking the selectable marker. concentrations were added to standard
the advantage of being independent of The pRS30x series of integrative YPD plates or liquid YPD medium
the presence of auxotrophic markers plasmids has some drawbacks. To after autoclaving and cooling to 60C:
in the yeast background. The common prevent multiple integrations, which 200 mg/L Geneticin Selective Reagent
vectors used for yeast manipulation could lead to overexpression of (G418; Invitrogen, La Jolla, CA, USA)
lack this advantage. They rely on the the inserted gene, we changed the (4) were added to select for kanMX4;
presence of an appropriate auxotrophic integration mechanism. The new 300 mg/L hygromycin B (InvivoGen,
marker. This led to the construction of plasmids do not create a tandem repeat Toulouse, France) were added to select
so-called designer deletion strains to of the auxotrophic marker with the for hphNT1; and 100 mg/L nourseo-
combine several auxotrophic markers integrated DNA sequence in the middle thricin (ClonNAT; Werner BioAgents,
into one single yeast strain (1,14,15). (3). Instead, they replace part of the Jena-Cospeda, Germany) were added
Due to experimental conditions, auxotrophic marker with the drug to select for natNT2. A sterile filtered
however, it is not always possible to resistance marker (kanMX4, natNT2, stock solution was used in the case
use one of these yeast strains, or the or hphNT1) and the DNA sequence of of ClonNAT, whereas Geneticin and
experiment may require growth on rich choice. hygromycin B were used as provided
media in which plasmid selection is not by the manufacturer.

Vol. 40, No. 1 (2006) BioTechniques 73


SHORT TECHNICAL REPORTS

Table 1. Centromeric and Episomal Plasmids drop-out powder (18) DNA [10 mg/mL salmon sperm DNA
lacking the appro- (Invitrogen) denatured at 100C for 10
Selection Replication
Name Marker Origin Control Digest
priate auxotrophic min and cooled on ice] were added.
marker. The appro- The cells were divided into appropriate
pRS41H hphNT1 ARS/CEN EcoRI: 1769; 3620 priate dominant drug aliquots and placed at -80C (no shock
pRS41K kanMX4 ARS/CEN HindIII: 1249; 3929 should be added after freezing). Usually, 10 L of competent
pRS41N natNT2 ARS/CEN XmaI: 1605; 3409 autoclaving of the cells were used for the transformation
pRS42H hphNT1 2 EcoRI: 1769; 4448
medium. of a centromeric or episomal plasmid
and 50 L of cells for the transfor-
pRS42K kanMX4 2 HindIII: 1249; 4757 mation of an integrative plasmid.
Yeast Transformation
pRS42N natNT2 2 XmaI: 1605; 4237 Thawed competent cells were added
Control digests to verify the plasmids can be made using the restric- The following to a sterile 1.5-mL tube containing the
tion enzymes indicated. The sizes of the resulting DNA fragments are protocol for yeast DNA (maximal 2 L plasmid DNA/10
given in base pairs. ARS, autonomously replicating sequence; CEN, transformation has L of cells). The suspension was mixed
centromeric sequence.
been previously well before a 6-fold volume of sterile
described (9). It is polyethylene glycol (PEG; 100 mM
The following media composition based on the lithium acetate method lithium acetate, 10 mM Tris-HCl, pH
was used for the double selection (20). Yeast cells were inoculated 8.0, 1 mM EDTA, 40% PEG 3350)
of an auxotrophic marker plasmid from a fresh pre-culture and grown was added. Cells were mixed again
together with a dominant drug resis- to a density of 0.50.7 A600 at 30C and incubated at room temperature
tance plasmid. The recipe was adapted (approximately 107 cells/mL) in YPD for approximately 30 min. Dimethyl
from the composition reported in the medium. Yeast cells were harvested sulfoxide (DMSO) was added to a
supplementary information (SGA by centrifugation (500 g for 3 min), final concentration of 10%. The cells
Analysis: Media) to Reference 19. The washed once with 0.10.5 volumes of were placed in a water bath at 42C
main change compared with standard sterile water, and once with 0.10.2 for 520 min (15 min works best with
synthetic complete (SC) medium was volumes of sterile SORB [100 mM most strains) and sedimented (23 min
the replacement of ammonium sulfate lithium acetate, 10 mM Tris-HCl, pH at 500 g). The cells were washed once
with monosodium glutamate as a 8.0, 1 mM EDTA/NaOH, pH 8.0, 1 M with YPD and resuspended in 3 mL of
nitrogen source. The medium contains sorbitol (special grade for molecular YPD. They were then incubated on a
20 g/L glucose, 1.7 g/L yeast nitrogen biology; Merck, Whitehouse Station, shaker for 46 h at 30C and spread on
base without ammonium sulfate and NJ, USA), adjusted with acetic acid to a plate with the appropriate dominant
amino acids (DifcoTM; BD, Franklin a pH 8.0]. The cells were resuspended drug selection marker.
Lakes, NJ, USA), 1 g/L monosodium in 360 L of sterile SORB per 50 mL Selection of drug resistance yeast
glutamic acid, and 2 g/L amino acid of cell culture and 40 L of carrier clones on plates often required replica

A B
Yeast origin of replication
(ARS/CEN or 2)

bla 3 end of multicloning site


kanMX4, Ampicilin integration site
hphNT1, resistance (HIS3, LEU2, or URA3)
Selection or natNT2
marker
Ampicilin
bla resistance Selection kanMX4,
integration site into the
marker hphNT1,
yeast genome
or natNT2

blue-white
selection
lacZ 5 end of integration site
(HIS3, LEU2, or URA3)

multicloning site
Figure 1. Features of the new episomal and centromeric plasmids (A) and the new integrative plasmids (B). The positions of the different selection
markers and multicloning and integration sites are indicated. Detailed maps are provided as supplementary material (Figures S1 and S2) that is available online
at www.BioTechniques.com. ARS, autonomously replicating sequence; CEN, centromeric sequence.

74 BioTechniques Vol. 40, No. 1 (2006)


plating on the same selective medium entire URA3 sequence in the plasmids performed before spreading the trans-
after 2 days at 30C due to the high pRS416 and pRS426. The kan, hph, formed cells onto selective medium.
background of transiently transformed and nat markers confer resistance to We used the plasmids mainly for gene
cells (9,12). Geneticin/G418 (6), hygromycin B, expression during sporulation. This
and nourseothricin (4), respectively. differentiation process requires growth
The three different resistance genes are phases in liquid-rich medium to obtain
RESULTS AND DISCUSSION under the control of the same promoter synchronized sporulating cultures. We
but differ in their terminators (5,6). tested the plasmids with two different
Centromeric and Episomal Plasmids Standard transformation procedures genes, SSP1 and SPO74. We found
with Dominant Selection Markers were used for the transformation into that both genes, when harbored on a
yeast. However, to allow expression new centromeric plasmid, are able to
Three different dominant drug resis- of the drug resistance, a nonse- complement the meiotic defects of the
tance markers were used to replace the lective adaptation phase of 46 h was corresponding null-gene mutants. Using
the episomal plasmids, we achieved
overexpression of several genes during
DNA of choice inserted meiosis. We did not observe any differ-
3 end of into the multicloning site ences in the new plasmids regarding
integration site
(HIS3, LEU2, or URA3)
transformation efficiency or plasmid
kanMX4, loss when compared with the common
hphNT1, pRS plasmids (unpublished observa-
or natNT2 tions). The plasmids have the same
5 end of integration
site (HIS3, LEU2, MCS as the other pRS plasmids (for
or URA3) restriction maps, see Supplementary
Figure S1). This provides many oppor-
Release of the integration cassette tunities to introduce DNA sequences.
by enzymatic digest Because the backbone is the same as
the other pRS plasmids, cloning of
inserts from already existing pRS-
based vectors into the new plasmids by
homologous recombination is possible.
Transformation An overview of the features of the new
into yeast centromeric and episomal plasmids is
shown in Figure 1A and in Table 1.
The drug resistance marker-based
centromeric or episomal plasmids
confer an advantage by allowing the
use of rich instead of synthetic media
Yeast chromosome
but are not limited to use in complete
medium. Combination with common
Integration at the
auxotrophic marker plasmids and
chosen integration site
growth in synthetic media is also
possible. Another advantage of the new
DNA of choice
plasmids is their lack of dependency
on a suitable auxotrophic marker in the
Yeast chromosome target strain. This enables one to work
dominant drug resistance gene with natural yeast isolates. Furthermore,
if all the available auxotrophic markers
Verify the correct in a yeast strain have been depleted due
integration by PCR to genetic manipulations, the use of
Primer1 the dominant drug resistance plasmids
Primer2 is still possible. During preparation of
this manuscript, we found that a similar
Yeast chromosome
approach was undertaken by Bardazzi
PCR product and Casalone (21). In this work, the
Figure 2. Step-by-step procedure to integrate DNA into the yeast genome using the newly created URA3 gene of plasmid pRS416 was
integrative plasmids. First, the DNA of choice has to be introduced into the multicloning site of one of replaced by either kanMX4 or LEU4*
the plasmids. This creates the cassette of drug resistance marker and DNA of choice flanked by regions (resistance to 5,5,5-trifluoro-DL-
homologous to HIS3, LEU2, or URA3. Then, the cassette is released from the vector by enzymatic digestion. Next,
the linearized DNA is transformed into yeast. There it integrates at the selected auxotrophic marker lo- leucine). The plasmids created in our
cus. After selection for the drug resistance, the yeast clones can be tested by PCR for correct insertion. work are an addition to the available
Vol. 40, No. 1 (2006) BioTechniques 75
SHORT TECHNICAL REPORTS

Table 2. Integrative Plasmids the cell. In this case, mixed populations


Selection Integration may appear that contain cells with and
Name Marker Site Control Digest without plasmid. All of these problems
are eliminated with the use of the new
pRS303H hphNT1 HIS3 HindIII+PvuII: 356; 958; 4565
set of plasmids because the mode of
pRS303K kanMX4 HIS3 HindIII+PvuII: 356; 958; 1056; 3283 integration is changed to a conventional
pRS303N natNT2 HIS3 HindIII+PvuII: 356; 958; 4182 deletion of the auxotrophic marker.
pRS305H hphNT1 LEU2 HindIII+PvuII: 3067; 3613 After the integration, the selection
can be omitted without risking the
pRS305K kanMX4 LEU2 HindIII+PvuII: 983; 2404; 3067
loss of the inserted DNA sequence.
pRS305N natNT2 LEU2 HindIII+PvuII: 3067; 3230 Therefore, the new plasmids allow a
pRS306H hphNT1 URA3 HindIII+PvuII: 356; 2659; 2765 more faithful integration of the desired
pRS306K kanMX4 URA3 HindIII+PvuII: 356; 983; 1556; 2659 DNA sequence into the yeast genome.
A similar vector had been previously
pRS306N natNT2 URA3 HindIII+PvuII: 356; 2382; 2659
constructed (23), but insertion was
Control digests to verify the plasmids can be made using the restriction enzymes indicated. The sizes of restricted to the HO locus. The new
the resulting DNA fragments are given in base pairs.
integrative plasmids provide more
possibilities and allow the integration
vectors. Centromeric and episomal by sequences homologous to the of several different DNA sequences
plasmids are now available with three respective target chromosomal locus. into the same yeast strain.
different dominant drug resistance Upon transformation in yeast, this Another method of DNA integration
markers. fragment is integrated into the genome has been developed recently, the so-
at the auxotrophic marker locus as in a called delitto perfetto (24,25). This
conventional gene deletion. A drawing method allows for the integration of
Yeast Integrative Plasmids with DNA at any site into the yeast genome.
Dominant Selection Markers of the mechanism is shown in Figure
2. This replacement mechanism repre- This is mainly important for the gener-
We created a set of yeast vectors sents the main difference between our ation of point mutations by transfor-
that can be used to integrate the DNA integrative plasmids and the pRS30x mation with oligonucleotides. Foreign
sequence of choice at three different plasmids, which form a tandem DNA can be inserted at any point
genomic loci, namely the URA3, LEU2, structure of the auxotrophic marker. into the yeast genome by using PCR
and HIS3. The corresponding mutants The transformation can be carried out in fragments with homologous flanking
are widely used as auxotrophic markers. the same way as the transformation of sequences. Nevertheless, this has some
Foreign DNA has been inserted at the centromeric and episomal vectors. disadvantages. The PCR fragments
these loci since the creation of the first Integration of two or three constructs at replace either a counter-selectable
integrative yeast plasmids (YIp) (1,22). different auxotrophic marker locations auxotrophic marker or have to include
The integrative plasmids with the is also possible. An overview of the a selectable marker. In the first case, a
drug resistance markers are based on features of the new integrative plasmids counter-selectable auxotrophic marker
pRS303, pRS305, and pRS306 (1). The is shown in Figure 1B and in Table 2. has to be introduced at the integration
MCS of these plasmids were removed, The commonly used integrative site. In the second case, the foreign
and the drug resistance genes, together plasmids of the pRS series have two DNA has to be inserted into a plasmid
with a new MCS, were inserted in the major disadvantages. First, trans- next to a suitable marker. In both
auxotrophic marker genes. Restriction formation often leads to multiple cases, the final yeast strain has to be
maps of the new integrative plasmids integrations of the plasmid (3). There checked for PCR errors by sequencing
can be found in the supplementary is no way to control this, and single the integrated DNA. The newly
material (Supplementary Figure S2). integration events can only be faith- created integrative plasmids offer
The restriction sites in the MCS are fully validated using Southern blot something similar to the second case.
marked in blue. Additional cloning sites analysis. Furthermore, the integration The integration is restricted to three
(in green) are included downstream of of a pRS30x plasmid generates a integration sites, but has the advantage
the selection marker. The integrative tandem structure of the auxotrophic that the sequencing of the integrated
plasmids can be used for transformation marker. This can lead to recombination DNA can be omitted.
as intact circular DNA if the appropriate and subsequent loss of the plasmid. Taken together, the new centromeric,
restriction site is missing (unpublished Moreover, selection for the integrated episomal, and integrative plasmids
observation), but using linearized plasmid does not prevent recombination enhance the flexibility of plasmid use
DNA enhances the efficiency consid- because the auxotropic marker can in yeast to a great extent. They allow a
erably. The plasmids were therefore be repaired during the process. Under free choice of growth medium, the use
cleaved before yeast transformation normal conditions, this recombination of yeast lacking a suitable auxotrophic
with suitable restriction enzymes to event is not very frequent. However, the marker, and enhance the fidelity of
release a fragment containing the DNA integrated plasmid may contain DNA plasmid use.
insert and the selection marker flanked that provides a selective disadvantage to
76 BioTechniques Vol. 40, No. 1 (2006)
ACKNOWLEDGMENTS 11.Sheff, M.A. and K.S. Thorn. 2004. Address correspondence to Christof Taxis,
Optimized cassettes for fluorescent protein EMBL, Cell Biology and Cell Biophysics
tagging in Saccharomyces cerevisiae. Yeast
The authors thank U. Kahl for her 21:661-670.
Unit, Meyerhofstr. 1, D-69117 Heidelberg,
assistance in cloning and C. Maeder, A. 12.Wach, A., A. Brachat, C. Alberti-Segui, Germany. e-mail: taxis@embl.de
Benjak, and D. Ditoro for their helpful C. Rebischung, and P. Philippsen. 1997.
comments on the manuscript. Heterologous HIS3 marker and GFP reporter
modules for PCR-targeting in Saccharomyces
To purchase reprints
cerevisiae. Yeast 13:1065-1075. of this article, contact
COMPETING INTERESTS 13.Gueldener, U., J. Heinisch, G.J. Koehler, D.
Voss, and J.H. Hegemann. 2002. A second Reprints@BioTechniques.com
STATEMENT
set of loxP marker cassettes for Cre-mediated
multiple gene knockouts in budding yeast.
The authors declare no competing Nucleic Acids Res. 30:e23.
interests. 14.Brachmann, C.B., A. Davies, G.J. Cost, E.
Caputo, J. Li, P. Hieter, and J.D. Boeke.
1998. Designer deletion strains derived from
REFERENCES Saccharomyces cerevisiae S288C: a useful
set of strains and plasmids for PCR-mediated
1. Sikorski, R.S. and P. Hieter. 1989. A system gene disruption and other applications. Yeast
of shuttle vectors and yeast host strains de- 14:115-132.
signed for efficient manipulation of DNA in 15.Replogle, K., L. Hovland, and D.H. Rivier.
Saccharomyces cerevisiae. Genetics 122:19-27. 1999. Designer deletion and prototrophic
2. Christianson, T.W., R.S. Sikorski, M. strains derived from Saccharomyces cerevi-
Dante, J.H. Shero, and P. Hieter. 1992. siae strain W303-1a. Yeast 15:1141-1149.
Multifunctional yeast high-copy-number 16.Ausubel, F.M., R.E. Kingston, F.G.
shuttle vectors. Gene 110:119-122. Seidman, K. Struhl, D.D. Moore, R. Brent,
3. Rothstein, R. 1991. Targeting, disruption, re- and F.A. Smith. 1995. Current Protocols in
placement, and allele rescue: integrative DNA Molecular Biology. John Wiley and Sons,
transformation in yeast. Methods Enzymol. New York.
194:281-301. 17.Knop, M. and E. Schiebel. 1998. Receptors
4. Goldstein, A.L. and J.H. McCusker. 1999. determine the cellular localization of a gamma-
Three new dominant drug resistance cassettes tubulin complex and thereby the site of micro-
for gene disruption in Saccharomyces cerevi- tubule formation. EMBO J. 17:3952-3967.
siae. Yeast 15:1541-1553. 18.Sherman, F. 2002. Getting started with yeast.
5. Janke, C., M.M. Magiera, N. Rathfelder, C. Methods Enzymol. 350:3-41.
Taxis, S. Reber, H. Maekawa, A. Moreno- 19.Tong, A.H., M. Evangelista, A.B. Parsons,
Borchart, G. Doenges, et al. 2004. A versa- H. Xu, G.D. Bader, N. Page, M. Robinson,
tile toolbox for PCR-based tagging of yeast S. Raghibizadeh, et al. 2001. Systematic
genes: new fluorescent proteins, more mark- genetic analysis with ordered arrays of yeast
ers and promoter substitution cassettes. Yeast deletion mutants. Science 294:2364-2368.
21:947-962. 20.Schiestl, R.H. and R.D. Gietz. 1989. High
6. Wach, A. 1996. PCR-synthesis of marker cas- efficiency transformation of intact yeast cells
settes with long flanking homology regions using single stranded nucleic acids as a carrier.
for gene disruptions in S. cerevisiae. Yeast Curr. Genet. 16:339-346.
12:259-265. 21.Bardazzi, I. and E. Casalone. 2004.
7. Wach, A., A. Brachat, R. Pohlmann, and P. Construction of two new vectors for transfor-
Philippsen. 1994. New heterologous modules mation of laboratory, natural and industrial
for classical or PCR-based gene disruptions Saccharomyces cerevisiae strains to trifluoro-
in Saccharomyces cerevisiae. Yeast 10:1793- leucine and G418 resistance. Folia Microbiol.
1808. (Praha) 49:534-538.
8. Gauss, R., M. Trautwein, T. Sommer, and 22.Parent, S.A., C.M. Fenimore, and K.A.
A. Spang. 2005. New modules for the repeat- Bostian. 1985. Vector systems for the expres-
ed internal and N-terminal epitope tagging sion, analysis and cloning of DNA sequences
of genes in Saccharomyces cerevisiae. Yeast in S. cerevisiae. Yeast 1:83-138.
22:1-12. 23.Voth, W.P., J.D. Richards, J.M. Shaw, and
9. Knop, M., K. Siegers, G. Pereira, W. D.J. Stillman. 2001. Yeast vectors for integration
Zachariae, B. Winsor, K. Nasmyth, and at the HO locus. Nucleic Acids Res. 29:E59.
E. Schiebel. 1999. Epitope tagging of yeast 24.Storici, F., L.K. Lewis, and M.A. Resnick.
genes using a PCR-based strategy: more tags 2001. In vivo site-directed mutagenesis using
and improved practical routines. Yeast 15:963-972. oligonucleotides. Nat. Biotechnol. 19:773-776.
10.Longtine, M.S., A. McKenzie 3rd, D.J. 25.Storici, F. and M.A. Resnick. 2003. Delitto
Demarini, N.G. Shah, A. Wach, A. Brachat, perfetto targeted mutagenesis in yeast with
P. Philippsen, and J.R. Pringle. 1998. oligonucleotides. Genet. Eng. (NY) 25:189-207.
Additional modules for versatile and eco-
nomical PCR-based gene deletion and modi-
fication in Saccharomyces cerevisiae. Yeast Received 2 June 2005; accepted
14:953-961. 29 August, 2005.

Vol. 40, No. 1 (2006) BioTechniques 77