Download Construction of a set of convenient saccharomyces cerevisiae

YEAST
VOL.
11: 53-55 (1995)
Construction of a Set of Convenient Saccharomyces
cerevisiae Strains that are Isogenic to S288C
FRED WINSTON*, CATHERINE DOLLARD AND STEPHANIE L. RICUPERO-HOVASSE
Department of Genetics, Harvard Medical School, 200 Longwood Avenue, Boston, M A 02115, USA
Received 2 May 1994; accepted 12 September 1994
A set of GAL2' yeast strains that are isogenic to strain S288C have been constructed. They contain non-reverting
mutations in genes commonly used for selection for recombinant plasmids. Strains from this collection are being
used for the European Union Yeast Genome Sequencing Programme. Representative strains from this collection
have been deposited with the ATCC.
K ~ WORDS
Y
- S288C;
isogenic; Saccharomyces cerevisiae.
Beginning with strain S288C (Mortimer, 1993;
Mortimer and Johnston, 1986), we have constructed an isogenic set of strains by gene replacement. The strains are GAL2' and contain
non-reverting mutations in URA3, HIS3, LEU2,
L YS2 and TRPl. They have already been distributed to many laboratories and have been used in
our laboratory for recent studies (for examples, see
Arndt et al., 1992 and Hirschhorn ei al., 1992). A
diploid derived from two of these strains is the
source of DNA for the European Union Yeast
Genome Sequencing Programme (Dujon, 1994;
Dujon rt al., 1994; Thierry and Dujon, 1992;
A. Thierry. C. Fairhead and B. Dujon, personal
communication).
Several plasmids were used to recombine
mutations into S288C. The plasmid used for recombining ura3-52 into the genome was pMRFW2
(Rose and Winston, 1984). The plasmids used for
recombining his3A200, leu2A1 and trplA63 into
the genome were YRp141his3A200, YRp15Ileu2Al
and YRp 14ltrplA63, respectively (Sikorski and
Hieter, 1989). The plasmid used for recombining
lvs2A202 into the genome was pJF210 (J. Fassler
and F. Winston, unpublished results). This plasmid is a derivative of plasmid pSL42-2 (Simchen
et a/., 1984) that contains a deletion of the XhoIHpaI fragment of L YS2. Plasmid YCp5O-HO was
used to convert mating type and was generously
provided by Rob Jensen and Ira Herskowitz. The
*Corresponding authoi
CCC 0749- 501)</95/010053-03
(
1995 bq John Wiley ti Sons Ltd
plasmid to transform strains to GAL,?' was pAAl
(generously provided by A. Antebe and G. R.
Fink). This plasmid, derived from a GALL?' clone
described by Tschopp et al. (1986), contains an
EcoRI-Hind111 fragment that contains GAL2' in
the plasmid YIp5.
All media and genetic methods are described
in Rose et al. (1990). Transformations were done
essentially as described by Schiestl and Gietz
(1 989).
The first step in the strain constructions was to
recombine ura3-52 into S288C. This was done as
follows: (1) 5 pg of plasmid pMRFW2 was digested with BamHI and SmaI; (2) the BamHISmaI fragment that contains ura3-52 was used to
transform strain S288C; (3) after the final step in
the transformation procedure (the incubation in
PEG), the cells were pelleted, resuspended in 5 ml
of YPD, and grown overnight; (4) the next day, the
cells were washed twice in water and resuspend
in 5 ml of water; and (5) dilutions of the cultures
were plated on 5-fluoro-orotic acid (5-FOA)
plates. Several 5-FOAR colonies were purified and
Southern analysis was performed to verify that
they contained the ura3-52 mutation. One of these
strains was saved and designated F Y 1.
The second step was to construct a GAL2'
derivative of FY1. This step was accomplished
using plasmid pAAl and two-step gene replacement (Scherer and Davis, 1979). One GAL2'
strain was saved and designated FY2 (Table 1). All
subsequent strains were derived from FY2.
54
Table 1. Yeast strains.
F. WINSTON I'T \L.
comparing the strains FY2, FY3 and S288C. All
chromosomes comigrated (data not shown).
Strain
Genotype
We tested the frequency at which strains FY1
and FY2 can be transformed by the plasmid
YCp50. Using the standard LiAc transformation
MA Ta ura3-52
FY2
method using denatured calf thymus DNA as the
FY3
M A Ta ura3-52
carrier (Schiestl and Gietz, 1989), transformation
FY4
MATa
MA Ta ura3-52 trpl A63
frequencies ranged from 1.4 x lo4 to 2.4 x lo4
FY7
FY8
M A Ta ura3-52 l.ys2A202
transformants/pg of YCp50 DNA. Using fresh
FYlO
MATa ura3-52 Ieu2AI
overnight cultures, the transformation frequencies
MA Ta ura3-52 his3A200
FY22
were substantially less, ranging from 350 to 1200
FY23
MATa ura3-52 trplA63 leu2A1
transformants/pg of YCp50 DNA. Experiments
FY67
MATa trplA63
with
other isogenic strains have shown that they
FY69
MATa leu2AI
can
be
transformed at the same approximate
FYI3
MA Ta his3A200 ura3-52
FY833
MATa his3A200 ura3-52 leu2A1 Ij*s2A202 frequencies (data not shown).
This set of strains is similar to a set previously
trp 1A63
FY834
M.4 Ta his3A200 ura3-52 leu2A1 lj)s2A202 described (Sikorski and Hieter, 1989). There are
trpl A63
two differences between the two sets of strains.
First, we began with S288C and made all constructions by gene replacement methods; in the previous
constructions, the beginning strain, YNN216, is
To obtain a MATa version of FY2, we trans- congenic to S288C (see Sikorski and Hieter. 1989).
formed FY2 with YCp5O-HO (generously pro- Second, the Galf phenotype of YNN216 is the
vided by Rob Jensen and Ira Herskowitz). The result of reversion of the ga/2 mutation found in
Uraf transformants were then streaked on 5-FOA S288C background, rather than a gene replaceplates and the single colonies were tested for their ment by the GAL2' gene (M. Carlson. personal
mating behavior. One MATa derivative was saved communication).
and designated FY3.
One significant advantage to using these strains
Auxotrophic markers for HIS3, LEU2, LYS2 is that a diploid made by mating strains FY23 and
and TRPl were recombined into strain FY2, re- FY73 (and designated stain FY 1679) was used as
placing the wild-type alleles, by two-step gene the source of DNA for a library that is being used
replacement. For each marker, we determined that for the European Union Yeast Genome Sequencthe auxotrophy segregated 2 : 2 in tetrads (data not ing Programme. This DNA has been or is curshown) and confirmed the gene replacement by rently being used for sequencing chromosomes
Southern analysis (data not shown). All strains VII, X, XI, XIV, XV and parts of IV and XI1 (B.
that contain combinations of these markers Dujon, personal communication; Dujon rt a/..
(strains with the FY designation; Table 1) were 1994). Strains derived from strain FY I679 have
constructed by genetic crosses.
also been used for several other genome-related
studies (for example, see Thierry and Dujon. 1993).
Struin cliuracterizations
To make these strains easily available. strains
To ensure that these strains were not disomic for FY23, FY67, FY69, FY73, FY833 and FY834
any chromosome, we crossed FY2 and FY3 by a have been deposited with the ATCC. The ATCC
set of strains to follow segregation of at least one numbers are as follows: FY23, 90840; FY67.
marker on each of the 16 yeast chromosomes. FY2 90841; FY69, 90842; FY73, 90843: FY833, 90844:
and FY3 were crossed by strains previously de- and FY834, 90845.
scribed by Gaber et al. (1983) and by Klapholz and
Esposito (1982). The results demonstrated that
every marker segregated 2 : 2 (data not shown). ACKNOWLEDGEMENTS
Therefore, each chromosome is monosomic in We thank Phil Hieter and Mark Rose tor helpful
these two strains. To address if there were any discussions during the Cold Spring Harbor Yeast
major chromosomal rearrangements that might Course that provided the motivation to make
not have been detected by these crosses, we exam- these strains. We thank Bernard Dujon for
ined the chromosomes by C H E F gel analysis, communicating unpublished results. This work
YEAST STRAINS ISOGENIC TO S288C
was supported by NIH grants GM32967 and
GM45720 to F.W.
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