YEAST VOL. 12: 667-672 (1996) A Simplified Method for the Repeated Replacement of Yeast Chromosomal Sequences with in vitro Mutations CHRISTOPHER SZENT-GYORGYI 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. KEY WORDS ~ CYH2"; counterselection; PCR. INTRODUCTION 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 668 C. SZENT-GYORGYI n r -2150 native locus -1500 I"-----... / I I * * / / \ \ / \ I 0 00 +161 CS36 11. '\ * * \ * target locus . . v . + z z* * z* 0 00 ..." A * 111.-3 : / ' 0 00 a-Arb, ... 2 direct replacement B ** * -=_ A ...' * i+ h 0 00 f .. . B' '..A I . * -e , I . p .\\ \,G \X LEU2 c- 'd CL .....,.. .., . I IV. . A indirect replacement .,.... ............ tandem intermediate I I 0 00 * * * -2150,-1750.-1500 +161,7536,+1447 lkb H I 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 allele. MATERIALS AND METHODS 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 MODIFIED GENE REPLACEMENT STRATEGY 669 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 1600 electroporation (Becker and Guarente, 1991). For 1600 all transformations, 1-5 pg linearized plasmid DNA and 10 pg of sonicated calf thymus carrier 740 660 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 530 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. 670 C . SZENT-GYORGYI 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 hybridization. RESULTS AND DISCUSSION 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 ~ are 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 below). 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 MODIFIED GENE REPLACEMENT STRATEGY 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 omitted. 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. 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