[ 19] TARGETING, DISRUPTION, REPLACEMENT, AND ALLELE RESCUE 281

added to the inositol starvation medium: adenine sulfate, uracil, L-argi- nine-HCl, L-histidine-HC1, L-methionine, L-tryptophan, each at 20 mg/ liter', L-isoleucine, L-leucine, L-lysine-HC1, L-tyrosine, each at 30 rag/liter; L-phenylalanine, 50 rag/liter; L-valine, 150 mg/fitvr, L-asparfic acid, L-glU- tamiC acid, each at 100 rag/liter; L-homoserine, L-threonine, each at 200 rag/liter; L-serine, 375 rag/liter. The last five nutrilites can often be omitted from the medium, since auxotrophs with these requirements are rare.

Synthetic prestarvation medium is inositol starvation medium plus 2000/~g of inositol. Starvation medium can also be made using Difco vitamin-free yeast nitrogen base (16.9 g/liter) but this provides 1% dextrose and also histidine, methionine, and tryptophan. YPD medium is 1% yeast extract, 2% peptone, and 2% dextrose. YPG medium is similar, but with 2% glycerol (v/v) replacing the dextrose.

[ 19] T a r g e t i n g , D i s r u p t i o n , R e p l a c e m e n t , a n d Al l e l e R e s c u e : I n t e g r a t i v e D N A T r a n s f o r m a t i o n in Y e a s t

By RODNEY ROTHSTEIN

Introduction

The ability to introduce exogenous DNA into microorganisms has been used extensively by investigators to manipulate the genomes of those organisms. ~ The availability of purified DNA fragments enabled yeast researchers in the late 1970s to introduce these fragments into yeast and for the first time achieve transformation reproducibly. 2,3 Hinnen, Hicks, and Fink 3 reported the transformation of yeast using a cloned LEU2 DNA fragment isolated by Ratzldn and Carbon. 4 They transformed a nonrevert- ing double mutation, 1eu2-3,112, and showed that yeast efficiently inte- grates circular DNA into the genome by a single homologous reciprocal exchange that results in a direct repeat of the target sequence. At the same time, Bcggs showed that LEU2 DNA, cloned in a plasmid that included sequences that allow the endogenous yeast plasmid, the 2-/~m circle, to replicate autonomously, also transform yeast. 2 In addition, chromosomal

S. A. Lacks, in "Genetic Recombination" (R. Kucherlapati and G. R. Smith, eds.), p. 43. American Society for Microbiology, Washington, D.C., 1989.

2 j. D. Bcggs, Nature (London) 275, 104 (1978). 3 A. Hinnen, J. B. Hicks, and G. R. Fink, Proc. Natl. Acad. Sci. U.S.A. 75, 1929 (1978). 4 B. Ratzkin and J. Carbon, Proc. Natl. Acad. Sci. U.S~. 74, 487 (1977).

cq~t~tt © t991 ~ Amdemk Pm~ htc. METHOD8 IN ENZYMOLOOY, VOL 194 All riJl~ofn~mxlu~in~ fonntum,,,,~L

282 toLmso MUrXNrS [19]

sequences that support autonomous replication (ARS) were discovered by their ability to cause an approximately 1000-fold increase in the frequency of transformation. 5 Manipulation of these autonomously replicating circu- lar plasmids led to the development of vectors that permitted the cloning by complementation, of any yeast selectable marker (see [ 14] this volume).

In the absence of ARS sequences, DNA transformed into yeast cells integrates into the genome exclusively by homologous recombination. Thus, sequences modified in vitro can be used to precisely replace the resident chromosomal copy of any cloned gene. 6 Homologous recombina- tion of transforming DNA can also be used to create null alleles by gene disruption of cloned yeast genes. TM The frequency of homologous recombi- nation after transformation can be stimulated by introducing a double- strand break within the yeast sequences on the plasmid. 9 Plasmids that contain more than one yeast homologous sequence can be integrated to the site of choice by introducing a double-strand break into that sequence on the plasmid.

The ability to direct integration of plasmids into any desired gene led to the development of methods to recover mutant chromosomal alleles by subsequently retrieving the sequence adjacent to the integrated plasmid as a circular plasmid in bacteria, t° Next, it was shown that gapped plasmid molecules repair the missing information using the resident chromosomal allele as template. H This permits the rescue of mutant alleles by genetic recombination with plasmids that contain autonomous replication se- quences. Methods to retrieve these plasmids in Escherichia coli are de- scribed in [21 ], this volume.

Utility of DNA Transformation

Transformation has been used to done genes by genetic complementa- tion, 12 to clone functional chromosomal components such as origins of replication, ~3 centromeres, ~4 and telomeres, ~5 and to clone functional sup-

K. StruM, D. T. Stinchcomb, S. Scberer, and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 76, 1035 (1979).

6 S. Scherer and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 76, 4951 (1979). * D. Shortle, J. E. Haber, and D. Botstein, Science 217, 371 (1982). 8 R. J. Rothstein, this series, Vol. 101, p. 202. 9 T. L. Orr-Weaver, J. W. Szostak, and R. J. Rothstein, Proc. Natl. Acad. Sci. U.S.A. 78,

6354 (1981). to j . I. Stiles, J. W. Szostak, A. T. Young, R. Wu, S. Consul, and F. Sherman, Cell (Cam-

bridge, Mass.) 25, 277 (1981). tl T. L. Orr-Weaver, J. W. Szostak, and R. J. Rothstein, this series, Vol. 101, p. 228. ,2 K. A. Nasmyth and S. I. Reed, Proc. Natl. Acad. Sci. U.S.A. 77, 2119 (1980). ]3 C. S. Chan and B. IC Tye, Proc. Natl. Acad. Sci. U.S.A. 77, 6329 (1980). 14 L. Clarke and J. Carbon, Nature (London) 287, 504 (1980). 15 j. W. Szostak and E. H. Blackburn, Cell (Cambridge, Mass.) 29, 245 (1982).

[19] TARGETINO, DISRUPTION, REPLACEMENT, AND ALLELE RESCUE 283

pressors identifying interacting genes. ~6 Once isolated, yeast chromosomal sequences can be manipulated in vitro and reintroduced into cells either on an autonomously replicating plasmid or at their normal chromosomal location to permit a functional analysis. Null mutations of any cloned gene can be created by introducing an in vitro-generated deletion into the genome at the precise chromosomal position of the gene. If a gene or region is difficult to score phenotypically, a genetic marker can be inserted adjacent to the gene or within the gene, resulting in an easily scored phenotypc. In addition, novel genotypcs can be created in which either new genes are introduced into regions to develop specific assays ~7 or novel chromosomal structures are created, such as shortened chromosomes use- ful for genetic analysis, ts A recently devised technique uses these methods to determine the physical map distance between a marker and its telo- mere. m9 Finally, the strategies described for yeast sequences can also be applied to foreign DNAs cloned in yeast on yeast artificial chromosomes. 2o

In this chapter, some of the basic strategies used for manipulating the yeast genome using exogenous DNA are described. Directed integration of plasmid sequences as well as several methods for gene disruption used to construct null mutations are discussed. Next, gene replacement strategies based on pop-in/pop-out recombination and fragment-mediated gene con- version are described. Finally, plasmid gap repair used to rescue chromo- somal alleles for DNA sequence and functional analysis is illustrated.

Materials

Yeast Strains and Media

Gene targeting and gene disruption can be performed in virtually any genetically marked yeast strain. The genetic markers for selection of trans- formation events in the host strain should exhibit low reversion frequen- cies (less than 10 -s). The most commonly employed auxotrophic markers are ura3, leu2, his3, and trpl, since double point mutations or low revert- ing alleles of these markers are available. In some cases, deletions have been engineered (e.g., his3-A200, 2~ trpl-A1 ~ ) that can be used to decrease

le I. Herskowitz, Nature (London) 329, 219 (1987). 17 j. W. Wallis, G. Chrebet, G. Brodsky, M. Rolfe, and R. Rothstein, Cell (Cambridge, Mass.)

58, 409 (1989). ~s A. W. Murray and J. W. Szostak, Nature (London) 305, 189 (1983). 19 D. Vollmth, R. W. Davis, C. Connelly, and P. Hieter, Proc. Natl. Acad. Sci. U.S.A. 85,

6027 (1988). 2o V. Pachnis, L. Pevny, R. Rothstein, and F. Costantini, Proc. Natl. Acad. Sci. U.S.A. 87,

5109 (1990). 21M. T. FasuUo and R. W. Davis, Mol. Cell. Biol. 8, 4370 (1988). ,2 R. S. Sikorski and P. Hieter, Genetics 122, 19 (1989).

284 MAKmO MUTANTS [ 19]

recombination between the incoming plasmid DNA and the chromosomal site. These methods are not limited to the markers described above. Any marker for which a suitable genetic selection exists ean be employed in targeting or gene disruption.

Standard genetic methods are used to grow yeast strains 23 (see [ 1 ] in this volume). Prototrophie transformants are seiected on synthetic medium lacking the nutrient for which the marked strain is auxotrophie. Recipes for drug-containing media [e.g., media with 5-fluoroorotic acid (5-FOA), cyeloheximide (CYH), or eanavanine (CAN)] are found in [20] in this volume.

Yeast Shuttle Vectors

There is no rule to follow in choosing the vector for use in targeted integration. Plasmids based on pBR322 ~ or pUC 2s are equally efficient in transformation. The yeast vector YIp5, 5 often used for pop-in/pop-out experiments, is shown in Fig. 1 along with some other useful cloning vectors for targeted integration. A series of multipurpose yeast vectors has also been described. 23

Restriction Enzyme Digestion

Restriction enzymes used for targeting can leave either a 5' overhang, a 3' overhang, or blunt ends on the DNA. No substantial difference in transformation effieiencies has been noted. Standard digestion buffers and reaction conditions recommended by the suppliers are followed. The pres- ence of residual restriction enzyme(s) in the transforming DNA does not appreciably inhibit or alter the transformation efficiencies.

Yeast Markers Used for Disruptions

Some commonly used selectable markers for disruption have been cloned in pUC-based vectors by John Hill ~ and are illustrated in Fig. 1. These vectors are available from our laboratory. Restriction sites that are unique or that lie outside the gene within the yeast DNA are underlined. These sites can be used to excise a functional fragment to use for gene disruption purposes.

23 F. Sherman, G. R. Fink, and J. B. Hicks, "Methods in Yeast Genetics." Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1986.

24 F. Bolivar, R. L. Rodriguez, P. J. Greene, M. C. Betlach, H. L. Heynecker, and H. W. Boyer, Gene 2, 95 (1977). C. Yanisch-Perron, J. Vieira, and J. M e ~ n ~ Gene 33, 103 (1985).

• s j . Hill, personal communication (1988).

[19] TARGETING, DISRUPTION, REPLACEMENT, AND ALLELE RESCUE 285

EcoRI C/al H/ndUl l~nd-II Nad

S~

H/txIIII

~ UNA3 ~ v Sa4 I / Xbal

Xmal ~ S BamHi

Kpnl EcoRI

Hindlll NOal Sphl Sphl

Nad Psd Nad Sail

Sa/~ Xbal I

EcoRV

I BsfEII

Sad BamHI EcoRI Xmd

Sad EcoRI

FIG. 1. Common yeast vectors. The four vectors shown can be used for ~ integra- tion or for extracting genetic markers for gene disruption. Yeast sequences are indicated by stippled fines. Restriction sites within the yeast fragments that can be used to extract a functional DNA fragment for gene disruption are underlined.

M e t h o d s

Integration by Homologous Recombination

Exogenous plasmid D N A that contains a yeast sequence, in the absence of A R S sequences, integrates at the homologous chromosomal locus after D N A transformation? Even plasmids that contain A R S sequences can integrate into the genome by homologous recombination. However, i f the

286 MAmNO MUTANTS [ 19]

plasmid contains centromere sequences, integration creates a dicentric chromosome, and the event is selected against. 27 Plasmid integration in yeast is used to genetically mark a region to determine if the cloned fragment of interest is tightly linked to the original complemented muta- tion. It is also the first step of the pop-in/pop-out replacement technique described below. 6 Last, it is used to insert a stable singe copy ofa gene at a unique chromosomal site. Plasmid integration is most etficienfly achieved by targeting the molecule with a double-strand break? However, plasmids that contain yeast fragments with lengths less than 250 base pairs (bp) cannot be targeted easily. 28

DNA Transformation with Circular Molecules: Nontargeted Integra- tion. In Fig. 2, a circular plasmid molecule is illustrated that contains two yeast sequences, A UXA for selection and YFG1 ("your favorite gene"). The recipient yeast strain is auxotrophic for auxA so that integratio~events can be selected. The recipient strain can be either auxotrophic or wild type for YFG1. After transformation, if there is no ARS on the plasmid, A UXA prototrophs arise from either a single reciprocal exchange between either A UXA or YFG1 and the corresponding homologous sequence in the chro- mosome (e.g., Fig. 2C 1) or a replacement of auxA with A UXA without any plasmid sequences integrated (likely by a gene conversion or a double crossover, Fig. 2C3). The relative ratio of integration versus replacement is fragment dependent. Colony hybridization using radioactively labeled bac- terial plasmid sequences as a probe distinguishes the integration events from replacements. 29 Genomic blots or genetic linkage can be used to determine into which homologous sequence the plasmid has integrated. Since the restriction enzyme sites surrounding the two regions are different (indicated by arrows in Fig. 2), a genomic blot digested with either of these enzymes and probed with radioactively labeled plasmid DNA will generate a unique pattern for each integration. 3° If the yeast fragment on the plas- mid contains a portion of a repetitive sequence such as a Ty element or a J sequence, 31 then integration into the homologous repeated sequences may occur, resulting in what appear to be random integration events. 32 For example, the PstI fragment of the LEU2 gene in YEpl 3 contains a portion ofa Ty element and its associated J sequence and was shown to integrate at genomic Ty elements. To avoid this problem, the HpaI-SalI LEU2 frag-

27 C. Mann and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 80, 228 (1983). 28 R. Rothstein, unpublished observations. 29 R. Rothstein, in "DNA Cloning: A Practical Approach" (D. M. Glover, ed.), p. 45. IRL

Press, Oxford, 1985. 3o T. Maniatis, E. F. Fritsch, and J. Sambrook, "Molecular Cloning: A Laboratory Manual."

Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1982. 3t j . D. Boeke, in "Mobile DNA" (D. Berg and M. Howe, eds.), p. 382. American Society for

Microbiology, Washington, D.C., 1989. 32 H. L. Klein and T. D. Petes, Mol. Cell. Biol. 4, 329 (1984).

A B

YFG UXA

AUXA

J - - T l ' ' ' ' ' ' ' ' ' I lyl~fl~ ' ' ' ' ' ' ' I ~ - L

C

(~) "-•"'"'"'"1• I YFGT AUXA

i '"""""~ j-- yfg; /

(2) - - - -~ '" '"""~l~rG, AUXA ~' ilOllllllll•:' l llll|llilll t "-L

(3) I I

FIG. 2. Targeted integration in yeast. (A) A circular plasmid that contains two yeast sequences, YFGI (barred area) and AUXA (dotted area), can pair with homologous chromo- somal sites, indicated as straight lines below the plasmid. The arrows surrounding each gene indicate restriction sites. The arrow on the plasmid and the upward arrows represent the same restriction site. The downward arrows represent unique chromosomal restriction sites that are not present on the plasmid. Note that the po~itions of the sites surrotmding the two genes are different. (B) Targeted integration into yfgl is achieved by creating a double-strand break, indicated by the space in the _d~hed line of the circular plasmid. The two ends of the linear molecule pair with the chromosomal sequence as indicated. The double-strand break stimu- lates recombination, and plasmid integration events occur at yfgl. (C) Three kinds of events are detected after selection for A UXA prototrophs. ( l ) A single crossover event at YFG1 leads to a direct repeat of YFGI flanking the plasmid. Genomic blots of DNA digested with a restr/ction enzyme that digests the plasmid once detect two bands with sizes characteristic for the integration site. Note that integration of the plasmid at auxA results in different restriction fragments (not shown). (2) A multiple integration event at YFGI leads to a third, plasmid-length, band after genomic blotting of DNA digested with a restriction enzyme that diges'ts the plasmid once. The number of plasmids/ntegrated can be determined by digesting genomic DNA with a restriction enzyme that does not digest the plasmid (downward arrow) and measuring the increased size of the parental band. (3) Replacement events occur at auxA when AUXA sequences from the plasmid replace the chromosomal mutant site, resulting in an A UXA prototroph.

288 Marm~o MUT~a~1"S [19]

ment in pUC-LEU2 (Fig. 1) is used since it does not have any repeated sequences.

Occasionally, multiple tandem integrations of the plasmid sequence OC~UI'. 9 These events can also be distinguished on genomic blots by digest- ing DNA from the transformant with a restriction enzyme that digests the plasmid once outside of the marker gene (indicated by the upward arrow). Hybridization with labeled plasmid DNA generates two bands for a single integration event. A multiple tandem array gives a third band equivalent in size to the plasmid (Fig. 2C2). The precise number of copies in the multiple tandem array can be determined by digesting with a restriction enzyme that fails to cut anywhere within the plasmid (indicated by the downward arrow) and measuring the increase in size of the parental fragment.

Transformation with Linear DNA Molecules: Targeted Integration. To ensure that the plasmid sequence integrates at the chromosomal location of interest, the integration event is targeted by introducing a double-strand break into the plasmid (Fig. 2B). 9 When the double-strand break is made within one of the two (or more) yeast sequences on a plasmid, the fre- quency of transformation increases 10-to 1000-fold compared to uncut circular plasmid without an ARS sequence. In addition, the double-strand break directs plasmid integration to the chromosomal region homologous to the cut sequence. An example of targeted integration is shown in Fig. 2B. To direct the integration event to yfgl, a unique double-strand break is introduced by restriction enzyme digestion within the YFG1 sequence on the plasmid. The best position for the double-strand break is determined by factors that may influence the frequency of integration, such as the length of homology adjacent to the double-strand break or the position of the break relative to a chromosomal mutant site (see next section). After transformation with the linearized DNA and selection for A UXA proto- trophs, plasmid integration events are readily detectable (Fig. 2C1, 2C2). In addition, at a lower frequency, replacement events at auxA may occur that have not integrated the plasmid sequence (Fig. 2C3). The distribution of these two kinds of events is dependent on factors such as the length of homology in the targeted sequence and the length of homology in the selectable marker and cannot be predicted a priori. As described above, integration events can be distinguished from replacements by performing yeast colony hybridization and genomic blots.

Variables that influence frequency of targeted integration. The role of the length of homology on the frequency of transformation has not been studied systematically. Anecdotal evidence suggests that longer homologies result in increased integration frequency. There is likely a minimum length of homology necessary for plasmid integration. In one case, cut sequences with 125 nucleotides of homology (37 nucleotides on one side and 88 on

[19] TARGETING, DISRUPTION, REPLACEMENT, AND ALLELE RESCUE 289

I X I . . . . ~"

o r

I X x I I X I

Fro. 3. C-ene conversion of a site adjacent to a double-strand break. When a double-strand break is introduced adjacent to the wild-type information on a fragment, there are two possible outcomes after integration into a region that contains a mutation (X). The plasmid integrates, creating a duplication of the target sequence, with one wild-type copy and one mutant copy. Alternatively, the wild-type information on the plasmid copy is converted to mutant, giving rise to a duplication in which both sides of the duplication 6~nking the plasmid sequence contain mutations. See text for further discussion.

the other) did not integrate whereas targeted integration was obtained with a sequence containing 250 nucleotides of homology (40 and 210). 2s

The natural variability of different genetic regions for recombination also affects integration frequencies. For example, the LEU2 region exhibits more than 100-fold lower integration frequencies when compared to the HIS3 region3 s These differences cannot be predicted in advance, so each case has to be tested independently. However, targeting generally increases significantly the probability of integration within a lyarticular sequence over that obtained with circular, uncut plasmid DNA.

The apparent frequency of integration is also influenced by the position of the mutant site on the chromosome relative to the cut site on the integrating plasmid. 33 During integration, mutant sequences from the chromosome can replace the wild-type information on the incoming DNA strand, resulting in direct repeats that each contain the mutant chromo- somal copy (Fig. 3). This is formally a gene conversion event, that is, the nonreciprocal transfer of genetic information. Orr-Weaver et al. showed that the frequency of gene conversion is highest when the break is closest to the mutant site. 33 Therefore, the position of the double-strand break affects the frequency at which wild-type information is converted to the mutant chromosomal sequence and affects the frequency of prototrophs recovered since a fraction of the integration events are lost to gene conversion.

The length of homology also influences the frequency of integration. To reduce the frequency of replacement events of the selectable marker and/or integration events that occur at exogenous sequences outside of the desired integration target, host strains that contain a deletion or rearrange-

33 T. L. Oft-Weaver, A. Nicolas, and J. W. Szostak, Mol. Cell. Biol. 8, 5292 (1988).

290 MAZrNo MUTANTS [ 19]

A 0 C

I I ',',, I I

FIo. 4. One-step gene disruption. (A) To make a mutation in YFG1 (thin line), (B) a seleetable marker (dotted area) is cloned into the middle of YFG1. The disrupted fragment is liberated from the parental plasmid using restriction enzymes (arrows). (C) The liberated DNA fragment is used to transform yeast. The homologous ends pair with the chromosome, and recombination results in a chromosomal gene replacement (D).

ment of the chromosomal copy of the selectable marker can be used. This reduces homology with the selectable marker and lowers the frequency of background replacements. For example, a plasmid linearized at YFG1, as illustrated in Fig. 2, integrates only at yfgl if the selectable marker se- quences (A UXA) have been deleted from the genome. The ura3-52 muta- tion is a Ty insertion that almost completely eliminates URA3 replacement events and reduces, but does not eliminate, integration events. ~

Gene Disruption Techniques

One-Step Gene Disruption. One-step gene disruption or replacement results in a genetically stable disruption since no direct repeats are left flanking the insertion site. s The method requires a cloned gene and a restriction map of the fragment to identify a restriction site(s) for inserting a selectable genetic marker (Fig. 4A). Two kinds of disruptions can be constructed. One, an insertion, results from inserting a genetic marker into a region at a single restriction site within the gene of interest. The other, an insertion-deletion, results in a deletion of all or a portion of the gene after the insertion. When creating a null mutation, it is best to delete as large a portion of the gene as possible to avoid the poss'bility that the insertion- disruption leads to a fortuitously functional fusion.

The selectable marker fragment used for the disruption is cloned into the gene of interest with sufficient homology adjacent to the insertion point

34 M. Rose and F. Winston, Mol. Gen. Genet. 193, 557 (1984).

[ 1 9] TARGETING, DISRUPTION, REPLACEMENT, AND ALLELE RESCUE 291

to permit homologous pairing with both sides of the chromosomal target sequence (Fig. 4B,C). The amount of DNA homology adjacent to the inserted gene should be greater than 500 bp whenever possible. Although no systematic study has been performed, the greater the length of homol- ogy, the more efficient the gene disruption. However, it is noteworthy that some gene disruptions have been successful with as few as 28 nucleotides of homology on one side of the insertion. 35

Next, the disrupted fragment is liberated from the plasmid vector by cutting with restriction enzymes that generate a linear fragment that is homologous to the chromosome at both of its ends (Fig. 4B,C). It is not always necessary to completely remove plasmid sequences from both ends of the linear fragment. In fact, up to 4000 nucleotides of plasmid sequence can be left on either end or on both ends as long as the unpaired end sequences are not homologous to any sequences in the yeast genome. These nonhomologous ends do not significantly lower the frequency of successful gene disruptions. Similarly, it is not necessary to purify the liberated fragment from the vector sequences before transformation unless sequences on the vector can recombine with other sequences in the ge- home such as other plasmid sequences in the recipient cell.

Disruptions work equally well with yeast transformation procedures based on spheroplast formation, 2,3 Li + ions, 36 or electroporation. 2s As a general rule 1 to 10/~g of plasmid (0.1-1 pmol o f a 15-kb plasmid) are used to transform l07 competent cells. Yields oftransformants vary from 1 to 1000 transformants/~g/107 cells.

It is important to verify a successful gene disruption by a genomic blot since occasionally the disrupted copy on the plasmid may integrate adja- cent to the wild-type genomic copy without replacing it. This leads to a duplication that still contains a functional gene and results in a misinter- pretation of the true null phenotype.

When the null phenotype is unknown, gene disruption experiments should be performed in diploid cells to maintain one wild-type copy after replacement. The diploid is sporulated, and tetrads containing the four products of meiosis are dissected to determine the phenotype of the disruption (Fig. 5). When all four spores survive, the spores containing the disrupting marker also contain the null allele. If only two spores survive and neither contains the marker used for the disruption, the gene is either essential for germination, essential for growth, or both. To distinguish between these possibilities, a wild-type copy of the gene is cloned onto a centromere plasmid that contains the URA3 gene and introduced into the

3s j. Strathern, personal communication (1982). 36 H. Ito, Y. Fukuda, IC Murata, and A. Kimura, ./. 8acteriol. 153, 163 (1983).

292 MAKING MUTANTS [ 19]

®

A I ' : . . . . . . . . ; I d issect @

I I te imds o r

©

© © Fxo. 5. Gene disruption in a diploid. When the disruption strategy shown in Fig. 4 is

applied to a diploid, one of the two chromosomes becomes disrupted. After sporulation and tetrad dissection, there are two possible outcomes: If the gene is nonessential, four spores survive, two containing the disrupted allele (dotted circle) and two wild-type spores (open circle). Alternatively, ff the disrupted gene is essential for germination, growth, or both, only the two wild-type spores survive.

heterozygous disruption strain by transformation. After sporulation and dissection, spores that contain the chromosomal gene disruption can be obtained since the wild-type plasmid copy can complement the defect. Next, plasmid loss is selected on 5-FOA medium which selects against the URA3-containing centromere plasmid. 37 If the gene is essential, cells con- raining the disrupted allele will fail to grow on 5-FOA medium. If the gene is essential only for germination, all of the cells will grow after selecting for plasmid loss.

Using standard gene disruption technology, a problem is encountered when multiple disruptions need to be analyzed, since the number of disruptions that can be created in a given strain is limited by the availabil- ity of useful selectable markers. Recently, a construct was designed to permit repeated disruptions without the loss of any selectable markers within the yeast strain. 38 This construct has a UIM3 gene cloned between duplicated copies of a fragment from the Salmonella hisG gene. This URA3 "cassette" is used to disrupt the cloned gene of interest, and the disruption is transferred to the chromosome as described above. The hisG direct repeats that flank the URA3 gene can recombine to leave a single hisG fragment disrupting the gene of interest. Recombinants selected on 5-FOA medium are ura3 auxotrophs, and thus the entire procedure can be repeated.

Transposon mutagenesis in bacteria has been used to create multiple independent insertions within a DNA fragment. Several useful sets of bacterial transposons have been engineered to contain yeast selectable

3~ j. D. Boeke, F. Lacrout¢, and G. R. Fink, Mol. Gen. Genet. 197,345 (1984). 38 E. Alani, L. Cao, and N. Kleclmcr, Genetics l l f b 54.1 (1987).

[ 19] TARGETING, DISRUPTION, REPLACEMENT, AND ALLELE RESCUE 293

marker genes. 39-41 One method is described in [22], this volume. In con- junction with these methods, the location of a gene on a cloned DNA fragment can be determined. Independent transposon-induced disruptions of a yeast fragment are transformed into yeast cells to saturate the region with insertion-mutations. If the null phenotype of the gene is unknown (and may be lethal), each independent transposition-disrupted fragment must be individually transformed into a diploid. To avoid aberrant events and ensure replacement at the chromosomal locus, the disruptions are introduced into a diploid that has been singly interrupted in the region of interest with a marker different from the one used for the transposon-me- diated disruptions. 42 After transformation and selection for the marker in the transposon, the colonies are screened for simultaneous loss of the first marker. Positive colonies are sporulated, and the four spores are dissected to assess the phenotype of the new transposon-generated disruptions.

Internal Fragment Disruption. A gene disruption can also be created by integrating a plasmid containing an internal segment of the gene into the homologous chromosomal copy of the gene. 7 The homologous reciprocal exchange between the internal fragment and the chromosome creates a disruption because, after integration, the two copies of the gene flanking the plasmid sequences are not full length (Fig. 6): one is truncated at the 3' end, and the other is truncated at the 5' end. The use of targeted integra- tion by cutting uniquely in the internal fragment increases the frequency of the disruption event. This method is useful when a convenient internal fragment is available (at least 350 bp). However, it is necessary to select continuously for the maintenance of the integrated plasmid sequence, otherwise spontaneous recombination results in loss of the plasmid at a frequency between 10 -4 and 10 --3, restoring the full-length gene. This high reversion frequency can often make it ditfieult to assess the precise pheno- type of the disruption.

Gene Replacement Techniques

In the previous section, techniques for gene disruption were described that lead to a complete loss of function of the gene of interest. The selectable marker itself was used to create the mutation, resulting in either

39 H. S. Seifert, E. Y. Chen, M. So, and F. Hetfron, Proc. Natl. Acad. Sci. U.S.A. 83, 735 (1986).

4o M. Snyder, S. Elledge, and R. W. Davis, Proc. Natl. Acad. Sci. U.S.A. 83, 730 (1986). 41 O. Huisman, W. Raymond, IC U. Froehlich, P. Errada, N. Kleckner, D. Botstein, and

M. A. Hoyt, Genetics 116, 191 (1987). 42 j. W. Wa~ W. L. Arthur, M. Roife, and R. Rothstein, in preparation.

294 MAKING MUTANTS [ 19]

I I I I I I

FIG. 6. Internal fragment disruption. An internal fragment of YFGI (thin line) is cloned into an integrating vector that contains a selectable marker (dotted area). Homologous recombination with the chromosomal locus results in a duplication that contains a mutated 3' fragment and a mutated 5' fragment; neither of which is functional.

an insertion or an insertion- deletion. In an exhaustive analysis of a gone, it is sometimes necessary to examine the phenotype of a large number of in vitro-generated constructs. The plasmid shuffle strategy described in [20], this volume, is one useful method for such an analysis. There are, however, occasions when the phenotype of a gone on a plasmid is different from the phenotype at its normal chromosomal location. 43'44 Methods for substitut- ing any kind of in vitro-constructed mutation back into the chromosome are described below.

Pop-In/Pop-Out Replacement. The pop-in/pop-out replacement method, developed by Scherer and Davis, 6 involves two steps: plasmid integration using the URA3 gone as a selectable marker and plasmid excision selecting against the URA3 gone. When the method was first described, the drug ureidosuccinic acid was used to select against the URA3 gone for the plasmid excision step. However, this selection does not work

43 j. Abraham, J. Feldman, K. A. Nasmyth, J. N. Strathern, A. J. Klar, J. R. Broach, and J. B. Hicks, Cold Spring Harbor Syrup. Quant. Biol. 47, 989 (1983).

44 A. H. Brand, L. Breeden, J. Abraham, R. Sternglanz, and K. Nasmyth, Cell (Cambridge, Mass.) 41, 41 (1985).

[19] TARGETING, DISRUPTION, REPLACEMENT, AND ALLELE RESCUE 295

A URA3

El U R A 3

I I ~ ' ~ - I v , i

C URA3

- +

D I X I

Fio. 7. Pop-in/pop-out allele replacement. (A) A mutation (X) is introduced in YFGI (thin line) and is cloned in an integrating vector that contains the URA3 selectable marker (dotted area). (B) Integration of the circular molecule results in direct repeats of YFG1 with one mutant copy and one wild-type copy. (C) Pairing of the homologous direct repeats and recombination can result in the loss of the plasmid sequence. This event is selected on 5-FOA-containing medium. (D) Crc~overs that occur on the appropriate side of the mutant site replace the wild-type chromosomal site with the mutant sequence.

296 MAKn~O MUT^m'S [1 9]

in all genetic backgrounds. Subsequently, the drug 5-FOA has been shown to work in virtually all genetic backgrounds tested. 37

The basic strategy of the pop-in/pop-out method is as follows: a specific alteration is introduced into the gene of interest cloned in a URA3-based integrating vector (e.g., YIp5 or pUC 18-URA3, see Fig. 7A). The alteration in the DNA can be a deletion, insertion, or a single base-pair change somewhere within the fragment. For example, a temperature-sensitive mutation or a suppressible mutation can be substituted for the wild-type copy as long as the mutant copy can be distinguished from the wild type (see below). Next, the plasmid is integrated into its chromosomal location by homologous recombination (Fig. 7B). This creates a duplication con- taining the wild-type copy and the mutant copy flanking the plasmid sequences. Finally, excision of the plasmid is selected using 5-FOA, and the 5-FOA-resistant colonies are screened for the mutant phenotype (see Fig. 7C,D).

The pop-in and pop-out events must occur on different sides of the alteration for the mutation to be retained after pop-out. It is best to leave as much homology on both sides of the mutant site as possible for the integration and excision events. For example, if the mutant site is asym- metrically positioned on the fragment, both the pop-in and the pop-out recombination events often occur on the same side of the mutation, and the altered site is not transferred to the chromosome. Targeted integration can be used to improve the probability of getting the pop-out to leave the mutant site in the chromosome. Whenever possible, the restriction site for targeting should be between the mutant site and the shortest stretch of homology. Although this does not guarantee that the pop-in crossover occurs in this position, it often does. This increases the probability that the pop-out crossover event will occur in the longer region of homology, resulting in a successful replacement. Finally, since the chromosomal se- quences can sometimes replace the mutant sequences in the integrating plasmid by gene conversion (e.g., see Fig. 3) the integration event itself can sometimes lead to loss of the mutation on the incoming plasmid. There- fore, it is best to have a method to detect the presence of the altered allele (e.g., a genomic blot) to ensure that, after the pop-in, the strain can generate the desired allele replacement by a pop-out.

Conditional mutations in essential genes, generated in vitro and screened in vivo by plasmid shuffling (see [20] in this volume for a com- plete description), can be introduced at their chromosomal location by using a modification of the pop-in/pop-out strate~. A diploid is disrupted with URA3 in one copy of the essential gene, deleting as much of the coding sequence as possible. The strain is transformed with the in vitro- generated mutant allele cloned into an integrating vector. Transformants

[19] TARGETING, DISRUPTION, REPLACEMENT, AND ALLEI.E RESCUE 297

A , I '" I

SUP4-o

B SUP¢o

c I IA I FIG. 8. Counterselecfion for allele replacement. (A) The gene of interest is first disrupted

with a counterselectable marker (SUP4-o in this example, dotted area), removing as much of the wild-type sequence of the gene as possible. (B) Linear DNA that contains the altered allele (X) is cotransformed into the recipient strain along with a selectable plasmid. The double crossover occurs nonselectively in approximately 0.1 to 5% of the transformants. (C) Trans- formants that have lost the counterselectable marker contain the replaced allele.

that integrate into the URA3-disrupted copy are identified by genomic blots or by genetic linkage. Haploid progeny containing the mutated allele linked to the disruption are isolated after sporulation and dissection. The conditional mutant is selected as a 5-FOA-resistant cell (ura3) that simulta- neously loses the integrating plasmid.

Use of Counterselectable Markers to Facilitate Direct Gene Replace- ment. Methods have been devised to facilitate repeated allele replacement at nonessential genes. First, a counterselectable marker is inserted at the chromosomal position of the gene (Fig. 8A). Next, linear DNA fragments containing the mutation of interest arc transformed into the strain, where they recombine with the resident disrupted region (Fig. 8B) and result in the loss of the counterselectable marker (Fig. 8C). The SUP4-o gene, the CAN1 gene, 45 or the CYH2 gene ~ can be used as counterselectable markers. To use the SUP4-o gene, the genetic background must contain an ochre-suppressible allele, such as canl-lO0. 47 To use the CANI gene, the strain may contain any allele of canl. ~ In either case, before the disruption with the counterselectable marker, the parent strain is resistant to cana- vanine, an arginine analog toxic to the cell. After introduction of the

45 W. Hoffmann, J. Biol. Chem. 260, 11831 (1985). N. F. Kaufer, H. M. Fried, W. F. Schwindinger, M. Jasin, and J. Warner, Nucleic Acids Res. U, 3123 (1983).

4t K. Nasmyth, Cell (Cambridge, Mass.) 42, 213 (1985). K. Struhl, Gene 26, 231 (1983).

298 MAmNG MUTANTS [1 9]

SUP4-o gene or the CAN1 gene at the locus of interest, the strain becomes sensitive to canavanine owing to restored function of the CAN1 gene, We arginine permease, and is ready for counterselection. After a successful replacement, the cell is again resistant to canavanine.

Replacements cannot be selected directly on canavanine-containing medium because the background level ofrevertants resistant to canavanine is too high ( - 10-~). Therefore, the most efficient way to substitute the mutated fragment into the chromosome is to cotransform the mutated linear molecule into the cell along with a circular plasmid that gives high-frequency transformation. The frequency of cotransformation varies from 0.1 to 5% of the total; after replica plating to canavanine medium, those transformants that have replaced the SUP4-o or CAN1 gene can grow.

A similar strategy applies for the CYH2 locus. 48 In this case, the starting strain is cycloheximide resistant (cyh2R), and the CYH2 wild-type gene is cloned into the gene of interest. Cotransformants are screened for cyclo- heximide resistance.

Allele Recovery

It is often desirable to recover mutant chromosomal alleles for DNA sequence analysis. Rather than constructing a library from each mutant strain, the most common way to retrieve a chromosomal mutation is based on the ability of DNA ends to promote homologous recombination and takes advantage of the efficient repair of gapped molecules. Using proce- dures similar to targeting, it was found that yeast recombines gapped linear molecules that are missing information from the targeted sequence.'l This requires that the two ends, homologous to the integration site, pair with the chromosome as illustrated in Fig. 9A. During the integration process, the gapped region is repaired using chromosomal sequences as template. Orr- Weaver and Szostak found that in the presence of an ARS sequence on the plasmid there are two possible outcomes for this repair event. 49 One results in the repair of the gap using chromosomal sequences as template, leading to an autonomously replicating plasmid that contains the mutant allele. The other is the integration of the plasmid after repair of the gap. Use of a centromere-based vector ensures that all of the gap repaired events are recovered as autonomously replicating plasmids. To rescue the mutant allele from such plasmids, total yeast DNA from the transformants is isolated (see [21 ] in this volume) and used to transform Escherichia coli.

This method has been modified to locate more precisely the region containing the mutant chromosomal allele. H For example, the plasmid

49 T. L. Oft-Weaver and J. W. Szostak, Proc. Natl. Acad. Sci. U.S.A. 80, 4417 (1983).

[19] TARGETING, DISRUYrloN, REPLACEMENT, AND ALLELI~ RESCUE 299

A

..... V I ,- V I I

- l X , . . . . , ~ ' ,* X I

B

I I I I I I I I I I Innl}( l l l l lnl l lnl l l l l l l l~-

IIIllnllJlllll l}~lllll l l]ll l n i le,-

FIG. 9. Allele rescue strategies. (A) The circular gapped plasmid contains an ar.~ sequence that permits it to replicate autonomously in yeast as a circle. When the gap spans the chromosomal mutant site (X), two kinds of events take place after repair of the gap. Either the gap is repaired and the plasmid integrates, resulting in a duplication of the mutant site on both sides of the vector, or, after gap repair, the circular plasmid ~plicates autonomously and contains the mutant chromosomal allele. In either case, the mutant allele can be rescued in E. coli as described in the text. (B) Integration of a plasmid adjac~t to the lp~ne of interest can also be used to rescue alleles. The integration event I__P~_ • to plasmid sequences juxtaposed to the allele of interest. ADpropriate restriction enzymes are used to cut either chromosomal DNA flanking the gene (downward arrows) or a restriction enzyme site that is present once in the plasmid and once near the gene (upward arrow). Cimdarization of these linear fresments by ligation and subsequent transformation in E. coli result in 8ene rescue as described in the t e x t .

300 ~AK~G MtrrANTS [ 19]

1 2 3

I X J

/or

I X I I ,,v I FIG. 10. Site mapping by plasmid gap repair. The position of mutant sites (X) can be

mapped using restriction enzymes to create different gaps. A site maps within a gap (2-3) when only mutant tran.~formants are recovered. If the mutant site maps outside of the gap (1 - 2), both wild-type and mutant plasmids are recovered. The frequency of wild-type gap-re- paired plasmids is dependent on the distance from the wild-type site on the plasmid to the border of the gap (as shown in Fig. 3).

illustrated in Fig. 10 can be gapped at separate locations. I f the chromo- somal muta t ion is contained within the gap, then all t ransformants will exhibit the mutan t phenotype. On the other hand, t ransformation with molecules gapped to the fight or the left o f the mutan t chromosomal site will result in a mixed populat ion o f t ransformants expressing either the mutan t or the wild-type phenotype o f the chromosomal allele. The mixed populat ion is due to the recovery o f some transformants that are mutant as a result o f gene conversion o f the wild-type information on the incoming

[19] TARGETING, DISRUPTION, REPLACEMENT, AND ALLELE RESCUE 301

plasmid DNA by the mutant chromosomal allele (see Fig. 3). A similar method has been described for mapping plasmid-borne mutations. 5°

Gap repair cannot be used for plasmids that contain repetitive se- quences since there are many alternative integration sites for the plasmid. A method that does not involve the use of gap repair can be used to rer~ver mutant sites from such sequences. It requires the integration of a plasmid adjacent to the allele of interest, as illustrated in Fig. 9B) ° After integra- tion, chromosomal DNA is isolated (see [I I] in this volume), and 100-300 ng is digested with a restriction enzyme that digests the DNA once near the end of the plasmid sequences and once near the end of the allele (Fig. 9B). The reaction is diluted to 1.0 ml and incubated with 1 m M ATP and 0.5- 1.0 unit of T4 DNA ligase at 15 ° overnight. Transformation of this ligation mix (diluted with 2 × transformation buffer) gives rise to between 10 and 50 tmnsformants, depending on the competence of the bacteria.

Summary

The methods described in this chapter permit the manipulation of virtually any cloned yeast chromosomal sequence by virtue of the fact that DNA transformed into yeast integrates into the chromosome by homolo- gous recombination. Furthermore, double-strand breaks in transforming DNA stimulate recombination and can be used to target integration events. This allows simple one-step gene disruption met.hods using yeast selectable markers. The availability of counterselectable markers makes it possible to replace chromosomal sequences with mutant alleles that cannot be directly selected. Finally, these same methods can be used to rescue chromosomal alleles on plasmids for subsequent molecular analysis.

Acknowledgments

I especially thank D. Shore for critical reading of the manuscript. I also thank A. Bailis, M. Fasullo, J. McDonald, and P. Vedander for their comments. I am an American Heart Association Established Investisator. This work was also supported by National Science Foundation Grant DCB 8703833, National Institutes of Health Grants GM34587 and CA21111, and grants from the Irma T. Hirschl Trust and the MacArthur Foundation.

so S. Kunes, H. Ma, K. Overbye, M. S. Fox, and D. Botstein, Genetics I15, 73 (1987).