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12: 667-672 (1996)
A Simplified Method for the Repeated Replacement of
Yeast Chromosomal Sequences with in vitro Mutations
Laboratory oj Cellulur und Developmental Biology, National Institute of Diabetes and Digestive and Kidney
Diseuses, National Institutes of Health, Bethesda, Maryland 20892, U.S. A .
Received 23 May 1995; accepted 9 November 1995
A strategy for gene replacement in Succhuromyces cerevisiae has been modified to facilitate the repeated substitution
of a chromosomal locus with in vitro generated variant sequences, so that the resulting locus contains only the
desired mutation and is free of extraneous vector DNA. The construction of an internally deleted chromosomal
target locus carrying the counterselectable CYH2' marker and a second positively selectable marker has been
simplified; the design of the locus has been altered to increase the frequency of authentic gene replacements obtained
upon the subsequent integration of in vitro mutated DNA. The modified chromosomal target locus is amenable to
replacement using either of two transformation protocols: (i) integration of a second positively selectable plasmid
carrying mutant sequences to form a tandem intermediate structure at the locus; upon counterselection on
cycloheximide, all vector sequence is excised to give the desired replacement at high frequency (>70%); (ii) single-step
integration of a linear segment of mutated genomic D N A by selection for cycloheximide resistance. A subsequent
screen for the loss of the positively selectable target locus marker detects the desired replacement at modest frequency
(22%). Polymerase chain reaction using multiple primers in a single amplification reaction is useful for monitoring
these variously modified chromosomal loci.
CYH2"; counterselection; PCR.
The efficient homologous recombination of DNA
in Saccharomyces cerevisiae has allowed the
development of powerful techniques for the
replacement of native chromosomal sequence with
exogenous DNA, so that the chromosome is free
of extraneous vector sequence and is altered only
by the desired mutation. In many studies it is
desirable to repeatedly replace the native DNA
with different variants that have been mutated
in vitro. The gene replacement strategy of Struhl
(1983) is especially well adapted to this purpose
because it entails the initial creation of a stable
chromosomal target locus deleted for much of the
gene of interest and marked by the cycloheximidesensitive gene CYH2' (Kaufer et al., 1983). This
target strain can then be repeatedly transformed
with integrating plasmids carrying alleles of the
gene that incorporate mutations mapping to
within those sequences deleted in the chromosome.
Integrants that subsequently excise the intervening
CCC 0749%503X/96/070667-06
0 1996 by John Wiley & Sons Ltd
vector sequences through recombination can be
selected by resistance to cycloheximide t o give at
modest frequency the desired gene replacement,
with no possibility of recovering the wild-type
allele as is the case with two-step transplacement
procedures (Winston et al., 1983).
In practice this gene replacement technique
suffers from a number of limitations. The twostep transplacement protocol that creates the
CYHZ'-marked chromosomal target locus can be
difficult and expensive. More important, this protocol suffers from a limitation in the design of the
target locus that greatly reduces the proportion
of desired gene replacements that are recovered
after the subsequent transformation with the
mutated allele. I present a simplified one-step
method for the construction of a modified target
locus that facilitates the repeated introduction of
mutated alleles by either indirect or direct transformation strategies; (i) integration of a plasmid
carrying the allele, followed by recombinative
excision of plasmid sequences; and (ii) direct
n r
native locus
* *
0 00
+161 CS36
* *
target locus
. .
v . + z z* * z*
0 00
: / '
0 00
direct replacement
0 00
I .
.....,.. ..,
. I
indirect replacement
.,.... ............
tandem intermediate
0 00
* *
Figure 1. Scheme for HSP82 gene replacement. DNA of the chromosomal HSP82 locus (I) is depicted as
unfilled bars; HSP82 DNA used to create the target locus (11) and introduce the mutated replacement alleles
(111, V) is respectively shown by light and dark grey bars. Locations of 5' (0)and 3' 1') junctions that
demarcate HSP82 DNA from these sources are also shown. Duplicated HSP82 DNA that can potentially
recombine to excise integrated vector DNA is delimited by labelled brackets. Numbers denote HSP82
coordinates in bp relative to the transcription start (Farrelly and Finkelstein, 1984). Labelled filled bars
depict selectable marker DNA; other plasmid DNA is shown by thin lines. The locations of PCR primers
used to assess the structure of integrants are also shown (a+e in 111). To create the stable target locus, the
0-deletion plasmid YIpCSG7 is integrated at the HSP82 locus (I) resulting in the net removal of HSP82
DNA between - 1500 and + 161 and the inclusion of the CYH2' allele at one flank (11). The arrangement
of the HSP82 inserts within the polylinker (thick line) of the 0-deletion plasmid is shown at the upper left;
the site at which this plasmid is linearized prior to integration is shown by the arrow. A second plasmid
carrying the in vitro generated mutation on a contiguous segment of HSP82 DNA from - 2150 to + 1447
(YIpCSG4 A130 is shown at the left side of 111; the deletion mutation is marked by A) is linearized with SpeI
at - 1750 to target integration to HSP82 sequences on the flank opposite to the CYHZ' marker (111).
Patching cells carrying the tandem integrants onto YPD plates containing cycloheximide gives rise to
papillants that have undergone recombination between duplicated HSP82 sequences (B to B') to excise all
intervening plasmid DNA. All such recombination events reconstruct the native HSPb'2 locus, which now
carries the desired mutation (IV). A second strategy directly replaces the target locus by transformation with
contiguous linear HSP82 DNA ( - 2150 to + 1447) and selecting for cycloheximide resistance (V).
integration of linear DNA carrying only the
The CYH2' gene ( - 317 to +1071; Kaufer et al.,
1983) was copied by polymerase chain reaction
(PCR) from plasmid pAB198 (gift of J. R.
Warner), in the process adding a 5' Sac1 and a
3' Sac11 restriction site. The PCR product was
inserted between the Sac1 and Sac11 sites in the
polylinker of pRS306 (Sikorski and Hieter, 1989)
to give plasmid YIpCSG1. DNA fragments cloned
from the HSP82 locus (Farrelly and Finkelstein,
1984; Szent-Gyorgyi, 1995) were then inserted into
the YIpCSGl polylinker to give a R-configuration
plasmid, YIpCSG7 (Sikorski and Hieter, 1989; see
Figure 1). A plasmid designed to reconstruct the
wild-type HSP82 locus (YIpCSG4) was created
by inserting a 3.6 kb HSP82 fragment ( - 2150
to +1447) between the XbaI and EcoRI sites of
the pRS305 polylinker (Sikorski and Hieter, 1989).
To construct a mutant HSP82 locus, the same
fragment carrying a 130 bp deletion between - 890
and - 1020 was inserted into pRS305 to give
YIpCSG4 A130.
YIpCSG7 was linearized at the XbaI polylinker
site and transformed into yeast strain S152 (a
~ $ 2 isolate
2 ~ of NKy274; Alani et al., 1987) by
electroporation (Becker and Guarente, 1991). For
all transformations, 1-5 pg linearized plasmid
DNA and 10 pg of sonicated calf thymus carrier
DNA were used per 40 pl cell suspension. Immediately after electroporation, 400 p1 ice-cold 1.0 M660
sorbitol was added, and 200 p1 of this suspension
was spread on uracil dropout plates (Rose et al.,
1990). Generally 30-500 colonies were obtained
per transformation. DNA was isolated from
URA3+ transformants (Hoffman and Winston,
1987) and the structure of the disrupted HSP82
locus was confirmed by Southern DNA hybridization (Church and Gilbert, 1984) and PCR (30
1 2 3 4 5
6 7 8 9 1 0
cycles; 60 s at 9 4 T , 60 s at 55"C, 90 s at 72°C; 10
Figure 2. Assessment of HSP82 gene replacement by PCR
min extension at 72°C after the last cycle in a using multiple primers. PCR products are labelled by size in bp
buffer [Innis et a/., 19881 containing 3 mM-Mg2+). and are derived from pairs of primers depicted in Figure 1 as
YIpCSG4 and YIpCSG4 A130 were linearized follows: 530, 660 bp, primers a, b; 740 bp, primers c, d; 1600 bp,
with SpeI, which cleaves at - 1750 of HSP82, and primers c, e. The left panel assesses HSP82 replacement by the
the recipient strain carrying the disrupted HSP82 standard protocol that maintains selection for both uracil and
leucine until plating on cycloheximide plates to select for
locus (CSGy20) was transformed by electropora- pop-outs. A mixture of four primers (a, b, c and e) was used to
tion. The structure of LEU2+ transformants was simultaneously assay for presence of wild-type (WT) or mutant
assessed by PCR, and cells carrying the tandem HSP82 sequence and the CYHZ' marker: lane 1, WT control
structure were patched onto buffered YPD (0.1 M- (structure I in Figure 1); lane 2, target locus (structure 11); lane
3, size standards; lane 4, replacement with HSP82 sequence
sodium succinate, pH 5.8) containing 10 pg/ml deleted
for 130 bp between - 890 and - 1020 (structure IV);
cycloheximide. Most patches displayed numerous lane 5 , replacement with WT HSP82 sequence. The right panel
papillae (> 100) after 2-3 days incubation at 30°C; assesses intermediate locus structures (variants of structure 111)
a low background of papillae (<5%) was some- observed after transformation with the second plasmid under
times seen in controls carrying the disrupted target leucine selection alone, but prior to selection of cycloheximideresistant papillants. A mixture of all five primers described
locus, presumably due to inactivating mutations above was used in the PCR assay. Most transformants display
within the dominant CYH2' marker or gene con- either the expected structure characterized by the 740 bp
plasmid junction PCR product (lane 9), or a structure that lacks
version by its chromosomal ~ y h 2counterpart.
Papillants were picked, assayed on replica plates the junction band (lane 6 ) , likely due to the excision of the
LIRA3 marker by recombination between repeated vector
for the loss of leucine and uracil prototrophy, and sequences
(mapping near primer c in Figure I). Transformants
DNA isolated from candidate pop-outs. The struc- harboring either of these structures readily papillate to give
ture of the HSP82 locus was assessed by PCR the desired pop-outs. Other LEU2+ candidate transformants
display different structures and do not papillate. An unaltered
using primers shown in Figure 2.
Disruption of the ARG4 and PHO3-PHO5 loci target locus (lane 8) presumably reflects gene conversion of the
chromosomal leu2 gene by the plasmid-borne LEU2 in the
was carried out as described using R-configuration absence of plasmid integration. Recombination between these
plasmids constructed by inserting appropriate mutant and WT LEU2 alleles apparently also can occur after
DNA fragments from these loci into YIpCSGl plasmid integration, leading to the retention of the plasmid
(T.-C. Wu and M. Lichten, personal communica- junction but removal of integrated HSP82 sequence as shown
lane 10. This recombination probably involves a chromotion). The disrupted target strains were then trans- in
somal rearrangement because the introduced LEU2 marker
formed as described using in vitro modified arg4 now segregates abnormally and independently of the CYH2
or pho5 linear DNA, but immediately following marker.
electroporation the transformation mixture was
diluted into 5.0 ml YPD containing 1.0 M-sorbitol.
This culture was grown overnight at 30°C, and
1OOyl of the culture was then plated onto
cycloheximide medium. Cycloheximide-resistant
colonies were replica-plated to medium lacking
uracil, and ura - colonies were picked for analysis of the avg4 or pho5 loci by Southern DNA
I have simplified several aspects of recipient strain
construction. The recipient strain must carry the
cycloheximide-resistant allele ( ~ y h 2 at
~ )the CYH2
locus, which in many cases necessitates the isolation of spontaneous cyh2" revertants. It is very
often not possible to obtain spontaneous ~ y h
isolates by directly plating cells onto YPD containing 1 or 10yg/ml cycloheximide (T.-C. Wu and
M. Lichten, C . Jozwik, personal communication,
and data not shown). One can enrich for
cycloheximide-resistant cells as follows. To a midlog phase culture (A600 0.5) growing in buffered
YPD (0.1 M-sodium succinate, pH 5.8) add 10 mg/
ml stock cycloheximide to give a final concentration of 2pg/ml. After 2 days further growth,
pellet the cells and resuspend in 1 ml water per
15 ml of original culture. Plate 200 p1 of the suspended cells per YPD plate (buffered as above)
containing 10 yg/ml cycloheximide. To ensure independent ~ y h 2isolates,
multiple original cultures are required. Authentic c ~ h 2strains
rendered sensitive to cycloheximide by transformation with the CYH2R allele described here (see
Materials and Methods). All tested cycloheximideresistant isolates segregated 2:2 (M. Lichten,
personal communication) and were ~ y h 2 ~ .
Creation of a chromosomal target locus by
two-step transplacement (Struhl, 1983) requires
the construction of an integrating URA3-marked
plasmid; its design is constrained by the availability of restriction enzyme digestion sites required to
splice the CYHZ' allele into the internally deleted
target gene, and required to linearize the construct
within target gene sequence to facilitate integration. After such a plasmid has been integrated, the
URA3' transformant must be counterselected
with 5-fluoroorotic acid (Boeke et al., 1987), an
expensive and somewhat mutagenic reagent, to
obtain a recombined chromosomal locus that
retains only the C Y1Y2~-disruptedtarget gene. To
circumvent these difficulties, a plasmid able to
stably disrupt non-essential chromosomal loci by a
single positively selectable integration event was
constructed by inserting the CYH2' gene at one
edge of the polylinker of pRS306 (Sikorski and
Hieter, 1989). It is relatively easy to ligate gene
segments that flank the intended chromosomal
deletion into this extensive polylinker in the required head-to-tail orientation (termed 'aintegration'; Sikorski and Hieter, 1989; see Figure
1). When linearized between these gene segments
and transformed into yeast under selection for
URA3+, this plasmid replaces much of the target
locus with vector DNA, positioning the CYH2'
marker immediately adjacent to native chromosomal sequence (see Figure 1). The position of
CYH2' is important to ensure that after the subsequent integration of a second plasmid bearing
2 the
~ mutation, selection of cycloheximide-resistant
cells gives only homologous recombinants between
target gene sequences at the flanks of the disrupted
locus (in Figure 1, between sequence B and B' of
the tandem intermediate structure), instead of
recombinants between duplicated gene or vector
sequences internal to the disrupted locus. The
retention of the URA3 marker in the disrupted
recipient locus facilitates crosses to transfer the
locus to different genetic backgrounds, provides
an important secondary screen used in both the
indirect and direct replacement methods, and also
reduces undesirable gene conversion events (see
The second integrating plasmid carries a
mutated segment of the target gene that spans
those native sequences deleted from the chromosome, overlapping into flanking chromosomal
sequences on both flanks of the deletion. There
must exist a unique restriction enzyme site within
this segment in sequences that are homologous to
the chromosomal flank opposite to the CYH2'
marker (such as the SpeI site in Figure 1). Transformation with plasmids linearized at this site
preferentially gives tandem vectors integrated at
the target locus (Figure 1, tandem intermediate). If
the host plasmids are related and are integrated
head-to-tail as in the example shown, under nonselective conditions recombination between the
vector backbones can lead to the frequent loss of
either the LEU2 or URA3 marker (but not both),
but these excisions do not compromise CYH2'based selection of desired chromosomal recombinants (see Figure 2). Although most LEU2+
transformants contain the recornbinable tandem
locus, structures that cannot produce pop-outs are
67 1
also observed (Figure 2). It is thus advisable to
maintain selection for both LEU2 and URA3 prior
to patching several independent LEU2+ transformants onto cycloheximide plates to counterselect for c ~ h 2papillants,
especially if an
assessment of the intermediate tandem structure is
Maintaining selection for these two markers also
has the important advantage of reducing undesirable gene conversion of the disrupted target allele
by the replacement allele (in Figure 1, conversion
of the interval A’-B’ by A-B) to a low relative
frequency (<lo%). In the procedure of Struhl, the
tandem intermediate chromosomal structure contains a single positively selectable URA3 marker
that resides external to the native target sequences
(to the left of A’-B‘ in Figure 1, corresponding
to the position of LEU2). The new allele carrying
the desired mutation thus can readily recombine
with its CYH2S-disrupted counterpart to produce
stable ~ y h strains
2 ~ that contain two copies of the
new allele, which bracket the intervening vector
sequence. These undesirable products may substantially outnumber authentic ~ y h gene
2 ~ replacements (85% of cycloheximide-resistant colonies
remain URA3+ and are presumed to represent this
gene conversion; Struhl, 1983).
Tandem chromosomal integrants have a
repetitive structure that can be difficult to assess
by Southern hybridization. An alternative means
of assessing structure is a PCR assay that detects
unique sequence or the junctions that demarcate
the integrated plasmids. A single PCR amplification reaction may use four to six primers that
give products of distinguishable sizes or alternatively anneal productively to only wild-type or
mutant sequence (Figure 2). In this example,
cycloheximide-resistant papillants that have undergone the desired recombination event lose two of
three PCR products concomitant with the loss of
leucine and uracil prototrophy.
The location of the CYH2’ gene within a doubly
marked target locus also facilitates single-step
replacement of the disrupting DNA, bypassing the
intermediate tandem chromosomal structure. In
this case, a mutated linear segment of native gene
DNA that overlaps both chromosomal flanks of
the target locus is used for transformation (see
Figure 1). Subsequent to transformation, cells
are grown overnight without selection in liquid
culture, and then plated onto cycloheximide plates.
C ~ h colonies
2 ~
are then screened by replica plating
onto medium lacking uracil to detect the loss of the
URA3 marker. This replacement technique has
been most thoroughly explored in experiments
with the ARG4 and PH03-PH05 loci (T.-C. Wu
and M. Lichten, personal communication); the
proportion of cycloheximide-resistant colonies
that are ura3- and contain the desired gene
replacement generally varies from about 2 to lo%,
but is occasionally as much as 95%). Similar
strategies that utilize direct selection during transformation to cycloheximide or 5-fluoroorotic acid
resistance have been respectively very inefficient or
unsuccessful (Struhl, 1983; Boeke et al., 1987). It is
unclear why there should be large differences in
success rate, especially between the protocols using
cycloheximide. One possibility is that the presence
of prokaryotic vector sequence in the target locus
(absent in the Struhl target) reduces the rate of
gene conversion of the CYH2’ marker by the
~ y h 2allele,
which otherwise may obscure the
relatively rare replacement events. It is also
possible that the transformation protocol used
here is highly efficient, so that integration events
are relatively numerous as compared to gene conversion events. Even in the case of the present
work, gene conversion a parently can account for
90% or more of the cyh2 isolates. The availability
of a secondary screen is clearly a prerequisite for
the practical implementation of this approach.
Different rates of integration due to differences in
homologous DNA overlaps or other chromosomal
locus characteristics could account for the
occasional high rate of target replacement, but it
should be noted that due to the outgrowth step
individual isolates may not be of independent
origin, and that differences in replacement
efficiency may be apparent rather than real. Singlestep replacement is potentially of great utility
because in principle one can directly transform
with linear DNA that has been synthesized entirely
in vitro by PCR mutagenesis methodologies.
I wish to thank Tzu-Chen Wu and Michael
Lichten for generously providing the cited data,
and Michael Lichten and Hugh Patterton for a
critical reading of the manuscript.
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