89NISR RESEARCH GRANT
2004 Young Investigator Research Grant

Establishment of “Highly Efficient” Transformation Methodof Saccharomyces cerevisiae Based on Molecular Basis for
Yeast’s Competence
Shigeyuki KAWAI
Department of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University
Research aims
Transformation of a yeast Saccharomyces cerevisiae isan indispensable method for the study of this good modelof the eucaryotic cell. An improvement of the transformability of the yeast cell has been required, since a transformability of yeast cell is lower by approximately 103fold than that of Escherichia coli. Understanding of the molecular mechanism for transformation of S. cerevisiae
would help to improve the transformation methods of not
only S. cerevisiae but also other eukaryotic cells. To understand the mechanism, transformability of all nonessential
gene mutants (about 5,000 strains) of S. cerevisiae was examined by natural transformation1 and the three mutants,
pde2, spf1, and pmr1 were found to show high transformability.2 The Pde2p is high-affinity cAMP phosphodiesterase.
In the pde2, intracellular level of cAMP is elevated. Therefore it has been predicted that elevated basal cAMP level
enhances transformability. Tpk1p, Tpk2p, and Tpk3p are
redundant catalytic subunits of protein kinase A (PKA),
which is a major cAMP target in the Ras-cAMP pathway.
PKA is a tetramer consisting of hetero- or homodimer ofthe catalytic subunits and homodimer of regulatory subunits encoded by BCY1. Taken together, it has been presumed that enhanced intracellular cAMP level increases
transformability through PKA.
This study was aimed to construct the S. cerevisiae
strains having the “highly efficient” transformability and tofind the transformation conditions giving “highly efficient”transformability.
Methods
The natural transformation method and lithium acetate(LiAc)/single-stranded carrier DNA (SS-DNA)/polyethylene glycol (PEG) method were conducted as described.1,3
Yeast cells were precultured overnight, transferred to fresh
YPD medium to reach A600 of 0.1, cultured to reach A600 of0.3 to 0.6, and then collected by centrifugation. In naturaltransformation method, the collected cells were suspended
in 20 m l TE (10 mM Tris-HCl, pH 8.0, and 1 mM EDTA),
and incubated with 20 m l plasmid DNA (0.5 m g YEp13 inTE) and 120 m l PEG4,000 (47% w/v in TE) for 1 h at 30°C,
and then for 5 min at 42°C. In LiAc/SS-DNA/PEG method,
the collected cells were suspended in LiAc/SS-DNA/PEG

solution (20 m l 0.5 m g YEp13, 36 m l 1 M LiAc in water,
50 m l 2 mg/ml SS-DNA in TE, and 240 m l 50% w/v
PEG3,350 in TE) and incubated for 40 min at 42°C. In bothmethods, after the cells were incubated, the cells were collected, washed once by 1 ml TE, and spread onto selective
SD solid medium (conventional method) or spread directly
onto selective SD solid medium without washing (directmethod). Just before spreading the cells onto SD selective
solid medium, numbers of viable cells were counted byspreading the diluted suspensions of the cells onto YPD
solid medium. Transformability was judged from the number of transformants and from the transformation frequency[the number of transformants/the number of viable cells].
Transformability is presented as an average value of morethan three independent determinations.
Gene disruption was performed by PCR-targeting as described.4 Strains used in this study are listed in Table 1.
Results
The first approach to construct the S. cerevisiae strains
having the “highly efficient” transformability was to identify the catalytic subunit of PKA concerning transformability. If one or two subunits inhibit transformability, deletingof the gene for the subunit was supposed to enhance transformability. If one or two subunits enhance transformability, an overexpression of the gene for the subunit was supposed to greatly enhance transformability. In order to validate this supposition, we examined the transformability ofthe following strains: (i) strains overexpressing each of
TPK1, TPK2, TPK3 (MK793, 794, 795), (ii) strains overexpressing BCY1 (the gene for regulatory subunit of PKA)
plus each of TPK1, TPK2, TPK3 (MK973, 974, 975), (iii)single mutants lacking each of BCY1, TPK1, TPK2, TPK3
(XPY1a , MK450, 451, 452), and (iv) double mutants lacking two of TPK1, TPK2, TPK3 (MB13, 23, 12) (Table 1).
However, transformability of these strains exhibited noobvious difference from that of isogenic wild type strain(WT) when transformability was examined by naturaltransformation method and LiAc/SS-DNA/PEG method(data not shown). We assumed that some effects of overexpressing of BCY1, TPK1, TPK2, TPK3 or deletions ofBCY1, TPK1, TPK2, TPK3 on transformability might bedetected through deletion of PDE2 from the tested strains.
Hence, we examined the transformability of the following

90

strains: (v) pde2 overexpressing each of TPK1, TPK2,TPK3 (MK979, 980, 981), (vi) pde2 overexpressing BCY1
plus each of TPK1, TPK2, TPK3 (MK940, 941, 942), (vii)
pde2 lacking each of TPK1, TPK2, TPK3 (MK871, 872,Fig. 1. Number of transformants and viable cells obtained by LiAc/SSDNA/PEG method (Li method) and natural transformation method (NTFmethod).
Averages of five (Li method) and three (NTF method) independent experiments are shown. YEp13 (0.5 m g) was used.
Table 1.

Fig. 2. Relative number of transformants obtained by conventional
method and direct method..
LiAc/SS-DNA/PEG method (Li method) and natural transformation
method (NTF method) were conducted. YEp13 (0.5 m g) was used. In eachmethod, the cells were spread on the selective medium and washing thecells once by 1 ml TE (conventional method) or without washing (directmethod). Averages of five (Li method) and three (NT method) independent
experiments are shown. Number of transformants ontained by NTF
method plus conventional method was relatively taken as 1.

S. cerevisiae strains used in this study.

Strain
BY4742
MK351
MK355
MK480
MK793
MK794
MK795
MK973
MK974
MK975
MK979
MK980
MK981
MK940
MK941
MK942
MK342
MK959
MK325
MK960
MK450
MK451
MK452
MK871
MK872
MK873
MK870
MK1341
MK1342
MK1343
W303-1A
MB13MB23MB12MK943
MK944
MK945
MK946
MYL40a
XPY1aGenotypeMATa leu2D 0 lys2D 0 ura3D 0 his3D 1MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 spf1D ::kanMX4
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 pde2D ::kanMX4
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 pmr1D ::kanMX4
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 [TPK1 in YEplac195]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 [TPK2 in YEplac195]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 [TPK3 in YEplac195]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 [TPK1 in YEplac195] [BCY1 in V056]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 [TPK2 in YEplac195] [BCY1 in V056]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 [TPK3 in YEplac195] [BCY1 in V056]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 pde2D ::kanMX4 [TPK1 in YEplac195]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 pde2D ::kanMX4 [TPK2 in YEplac195]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 pde2D ::kanMX4 [TPK3 in YEplac195]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 pde2D ::kanMX4 [TPK1 in YEplac195] [BCY1 in V056]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 pde2D ::kanMX4 [TPK2 in YEplac195] [BCY1 in V056]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 pde2D ::kanMX4 [TPK3 in YEplac195] [BCY1 in V056]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 [YEplac195]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 [YEplac195] [V056]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 pde2D ::kanMX4 [YEplac195]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 pde2D ::kanMX4 [YEplac195] [V056]
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 tpk1D ::kanMX4
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 tpk2D ::kanMX4
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 tpk3D ::kanMX4
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 tpk1D ::kanMX4 pde2::URA3
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 tpk2D ::kanMX4 pde2::URA3
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 tpk3D ::kanMX4 pde2::URA3
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 pde2::URA3 spf1D ::kanMX4
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 pde2::URA3 pmr1D ::kanMX4
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 pmr1D ::kanMX4 spf1D ::HIS3MX6
MATa leu2D 0 lys2D 0 ura3D 0 his3D 1 pde2::URA3 pmr1D kanMX4 spf1D ::HIS3MX6
MATa ade2-1 his3-11,15 lue2-3,112 trp1-1 ura3-1 can1-100
MATa ade2-1 his3-11,15 lue2-3,112 trp1-1 ura3-1 can1-100 tpk1::LUE2 tpk3::URA3
MATa ade2-1 his3-11,15 lue2-3,112 trp1-1 ura3-1 can1-100 tpk2::HIS3 tpk3::URA3
MATa ade2-1 his3-11,15 lue2-3,112 trp1-1 ura3-1 can1-100 tpk1::LUE2 tpk2::HIS3
MATa ade2-1 his3-11,15 lue2-3,112 trp1-1 ura3-1 can1-100 pde2D ::kanMX6
MATa ade2-1 his3-11,15 lue2-3,112 trp1-1 ura3-1 can1-100 pde2D ::kanMX6 tpk1::LUE2 tpk3::URA3
MATa ade2-1 his3-11,15 lue2-3,112 trp1-1 ura3-1 can1-100 pde2D ::kanMX6 tpk2::HIS3 tpk3::URA3
MATa ade2-1 his3-11,15 lue2-3,112 trp1-1 ura3-1 can1-100 pde2D ::kanMX6 tpk1::LUE2 tpk2::HIS3
MATa ura3-52
MATa ura3-52 bcy1::kanMX2

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91873), and (viii) pde2 lacking two of TPK1, TPK2, TPK3
(MK944, 945, 946). However, transformability of thesestrains exhibited no obvious difference from that of pde2.
Then, we decided to examine the transformation conditions giving “highly efficient” transformability by using
mutants with the high transformability, i.e., pde2, spf1, andpmr1. LiAc/SS-DNA/PEG method gave higher transformability of the mutants than natural transformation method(Fig. 1).
The high transformability mutants showed higher transformability than WT. We have recently found that (i) plasmid DNA is bound to the region around cell surface duringtransformation process and (ii) washing the cells, on whichplasmid DNA is bound, results in removal of the boundplasmid DNA from the cell and in decrease of transformability (Tuan et al., unpublished data). The yeast cells are
washed just before spreading on the selective solid medium
in conventional methods.1,3) Based on our recent finding,transformability obtained by the conventional method wascompared with that obtained without washing, i.e. that obtained by spreading the cell directly (by the direct method)(Fig. 2).
Transformability of the cells tested (WT, pde2, spf1,pmr1) was enhanced by the direct method. Accordingly, before spreading the cells on the selective solid medium toobtain suitable number of colonies for counting, dilution of
the cells in 35% PEG4,000 gave higher transformability
than that in usual buffer (e.g. TE) (data not shown).
We expected that the double mutants for PDE2, SPF1,and PMR1 show higher transformability than single mutants and that the triple mutant exhibits the much higher
transformability. Hence the mutants were constructed(MK870, MK1341, 1342, 1343; Table 1) and the transformability of the mutants was examined. However, transformability was not enhanced in the double and triple mu-tants.
Conclusion
In order to obtain the highly efficient transformability, wepoint out the followings. First, disruption of “one”, but not
two or three of PDE2, SPF1, and PMR1 in the yeast cellwould improve the transformability. Second, LiAc/SSDNA/PEG method gives higher transformability of pde2,spf1, and pmr1 than WT and those obtained by naturaltransformation method. Third, the direct method, in which
the cells are not washed before spreading on the selective
solid medium, gives higher transformability than that obtained by conventional method in which the cells are“washed”. Finally, when the cells are required to be diluted
before spreading the cells on the selective solid medium,the cells should be diluted in 35% PEG4,000, but not inusual buffer (e.g. TE).
References1.

Hayama, Y., Y. Fukuda, S. Kawai, W. Hashimoto, and K. Murata.
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Fukuda, and K. Murata. 2004. Molecular insights on DNA delivery
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3. Gietz, R. D., and R. A. Woods. 2002. Transformation of yeast bylithium acetate/single-stranded carrier DNA/polyethylene glycol
method. Methods Enzymol. 350: 87–96.
4. Shi, F., S. Kawai, S. Mori, E. Kono, and K. Murata. 2005. Identification of ATP-NADH kinase isozymes and their contribution to supplyof NADP(H) in Saccharomyces cerevisiae. FEBS J., 272: 3337–3349.