CHAPTER 9

Cloning in Saccharomycescerevisiae and other fungi

Introduction

The analysis of eukaryotic DNA sequences has beenfacilitated by the ease with which DNA from eukary-otes can be cloned in prokaryotes, using the vectorsdescribed in previous chapters. Such cloned sequencescan be obtained easily in large amounts and can bealtered in vivo by bacterial genetic techniques and invitro by specific enzyme modifications. To determinethe effects of these experimentally induced changeson the function and expression of eukaryotic genes,the rearranged sequences must be taken out of thebacteria in which they were cloned and reintroducedinto a eukaryotic organism. Despite the overall unityof biochemistry, there are many functions commonto eukaryotic cells which are absent from prokary-otes, e.g. localization of ATP-generating systems tomitochondria, association of DNA with histones,mitosis and meiosis, and obligate differentiation ofcells. The genetic control of such functions must beassessed in a eukaryotic environment.

Ideally these eukaryotic genes should be reintro-duced into the organism from which they wereobtained. In this chapter we shall discuss the poten-tial for cloning these genes in Saccharomyces cere-visiae and other fungi and in later chapters we shallconsider methods for cloning in animal and plantcells. It should be borne in mind that yeast cells aremuch easier to grow and manipulate than plant andanimal cells. Fortunately, the cellular biochemistryand regulation of yeast are very like those of highereukaryotes. For example, signal transduction andtranscription regulation by mammalian steroidreceptors can be mimicked in strains of S. cerevisiaeexpressing receptor sequences (Metzger et al. 1988,Schena & Yamamoto 1988). There are many yeasthomologues of human genes, e.g. those involved incell division. Thus yeast can be a very good surrog-ate host for studying the structure and function ofeukaryotic gene products.

Introducing DNA into fungi

Like Escherichia coli, fungi are not naturally trans-formable and artificial means have to be used forintroducing foreign DNA. One method involves theuse of spheroplasts (i.e. wall-less cells) and was firstdeveloped for S. cerevisiae (Hinnen et al. 1978). Inthis method, the cell wall is removed enzymically andthe resulting spheroplasts are fused with ethyleneglycol in the presence of DNA and CaCl2. Thespheroplasts are then allowed to generate new cellwalls in a stabilizing medium containing 3% agar.This latter step makes subsequent retrieval of cellsinconvenient. Electroporation provides a simpler andmore convenient alternative to the use of sphero-plasts. Cells transformed by electroporation can beselected on the surface of solid media, thus facilitat-ing subsequent manipulation. Both the spheroplasttechnique and electroporation have been applied toa wide range of yeasts and filamentous fungi.

DNA can also be introduced into yeasts andfilamentous fungi by conjugation. Heinemann andSprague (1989) and Sikorski et al. (1990) found thatenterobacterial plasmids, such as R751 (IncPβ) andF (IncF), could facilitate plasmid transfer from E. colito S. cerevisiae and Schizosaccharomyces pombe. Thebacterial plant pathogen Agrobacterium tumefacienscontains a large plasmid, the Ti plasmid, and part of this plasmid (the transfered DNA (T-DNA)) can beconjugally transferred to protoplasts of S. cerevisiae(Bundock et al. 1995) and a range of filamentousfungi (De Groot et al. 1998). T-DNA can also betransferred to hyphae and conidia.

The fate of DNA introduced into fungi

In the original experiments on transformation of S.cerevisiae, Hinnen et al. (1978) transformed a leucineauxotroph with the plasmid pYeLeu 10. This plasmidis a hybrid composed of the enterobacterial plasmid

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Cloning in S. cerevisiae and other fungi 157

ColE1 and a segment of yeast DNA containing theLEU2+ gene and is unable to replicate in yeast.Analysis of the transformants showed that in someof them there had been reciprocal recombinationbetween the incoming LEU2+ and the recipientLeu2− alleles. In the majority of the transformants,ColE1 DNA was also present and genetic analysisshowed that in some of them the LEU2+ allele wasclosely linked to the original Leu2− allele, whereas in the remaining ones the LEU2+ allele was on a different chromosome.

The results described above can be confirmed byrestriction-endonuclease analysis, since pYeLeu 10contains no cleavage sites for HindIII. When DNAfrom the Leu2− parent was digested with endonucle-ase HindIII and electrophoresed in agarose, multipleDNA fragments were observed but only one of thesehybridized with DNA from pYeLeu 10. With thetransformants in which the Leu2− and LEU2+ alleleswere linked, only a single fragment of DNA hybridizedto pYeLeu 10, but this had an increased size, con-sistent with the insertion of a complete pYeLeu 10molecule into the original fragment. These data areconsistent with there being a tandem duplication ofthe Leu2 region of the chromosome (Fig. 9.1). Withthe remaining transformants, two DNA fragmentsthat hybridized to pYeLeu 10 could be found on electrophoresis. One fragment corresponded to thefragment seen with DNA from the recipient cells,the other to the plasmid genome which had been

inserted in another chromosome (see Fig. 10.1). Theseresults represented the first unambiguous demon-stration that foreign DNA, in this case cloned ColE1DNA, can integrate into the genome of a eukaryote.A plasmid such as pYeLeu 10 which can do this isknown as a yeast integrating plasmid (YIp).

During transformation, the integration of exogen-ous DNA can occur by recombination with a homo-logous or an unrelated sequence. In most cases,non-homologous integration is more common thanhomologous recombination (Fincham 1989), butthis is not so in S. cerevisiae (Schiestl & Petes 1991).In the experiments of Hinnen et al. (1978) describedabove, sequences of the yeast retrotransposon Ty2were probably responsible for the integration of the plasmid in novel locations of the genome, i.e. the ‘illegitimate’ recombinants were the result ofhomologous crossovers within a repeated element(Kudla & Nicolas 1992). Based on a similar prin-ciple, a novel vector has been constructed by Kudlaand Nicolas (1992) which allows integration of a cloned DNA sequence at different sites in thegenome. This feature is provided by the inclusion in the vector of a repeated yeast sigma sequence present in approximately 20–30 copies per genomeand spread over most or all of the 16 chromosomes.

When T-DNA from the Ti plasmid of Agrobacteriumis transferred to yeast, it too will insert in differentparts of the genome by illegitimate recombination(Bundock & Hooykaas 1996).

Direction of migration

Electrophoretic separationof HindIII generatedfragments which hybridizewith pYeleu 10

Chromosome structure oftransformants and recipient

Recipient

HindIII HindIII

Leu 2–

Leu 2+

Leu 2– pYeleu10

Leu 2–

pYeleu10

Transformants

Fig. 9.1 Analysis of yeast transformants.(See text for details.)

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158 CHAPTER 9

Schiestl and Petes (1991) developed a method forforcing illegitimate recombination by transformingyeast with BamH1-generated fragments in the pres-ence of the BamH1 enzyme. Not only did this increasethe frequency of transformants but the transformantswhich were obtained had the exogenous DNA integ-rated into genomic BamH1 sites. This technique,which is sometimes referred to as restriction-enzyme-mediated integration (REMI), has been extended toother fungi, such as Cochliobolus (Lu et al. 1994),Ustilago (Bolker et al. 1995) and Aspergillus (Sanchezet al. 1998).

Plasmid vectors for use in fungi

If the heterologous DNA introduced into fungi is tobe maintained in an extrachromosomal state thenplasmid vectors are required which are capable ofreplicating in the fungal host. Four types of plasmidvector have been developed: yeast episomal plasmids(YEps), yeast replicating plasmids (YRps), yeast centromere plasmids (YCps) and yeast artificialchromosomes (YACs). All of them have features incommon. First, they all contain unique target sitesfor a number of restriction endonucleases. Secondly,they can all replicate in E. coli, often at high copynumber. This is important, because for many exper-iments it is necessary to amplify the vector DNA in E. coli before transformation of the ultimate yeastrecipient. Finally, they all employ markers that can be selected readily in yeast and which will oftencomplement the corresponding mutations in E. colias well. The four most widely used markers are His3,Leu2, Trp1 and Ura3. Mutations in the cognate chro-mosomal markers are recessive, and non-revertingmutants are available. Two yeast selectable mark-ers, Ura3 and Lys2, have the advantage of offeringboth positive and negative selection. Positive selec-tion is for complementation of auxotrophy. Negativeselection is for ability to grow on medium containinga compound that inhibits the growth of cells ex-pressing the wild-type function. In the case of Ura3,it is 5-fluoro-orotic acid (Boeke et al. 1984) and for Lys2 it is α-aminoadipate (Chatoo et al. 1979).These inhibitors permit the ready selection of thoserare cells which have undergone a recombination orloss event to remove the plasmid DNA sequences.The Lys2 gene is not utilized frequently because it is

large and contains sites within the coding sequencefor many of the commonly used restriction sites.

Yeast episomal plasmids

YEps were first constructed by Beggs (1978) by recombining an E. coli cloning vector with the nat-urally occurring yeast 2 µm plasmid. This plasmid is6.3 kb in size, has a copy number of 50–100 perhaploid cell and has no known function. A repres-entative YEp is shown in Fig. 9.2.

Yeast replicating plasmids

YRps were initially constructed by Struhl et al. (1979).They isolated chromosomal fragments of DNA whichcarry sequences that enable E. coli vectors to replic-ate in yeast cells. Such sequences are known as ars (autonomously replicating sequence). An ars isquite different from a centromere: the former acts as an origin of replication (Palzkill & Newlon 1988,Huang & Kowalski 1993), whereas the latter isinvolved in chromosome segregation.

Aat II

Ase IAhd I

Tth111I

Pvu IIBsaB I

BspE I

Eag IPshA I

Sal IEcoN I

Sph ISgrA I

BamH INhe I

Sma I/Xma I

Api I/Bsp1201 I

Nco I

Sbf I

Cla I -BspD I

Blp I

Hpa I

SnaB I

Xba I

Mfe IBcl I

YEp 24

ORI

amp

Tc

ura3

2µ circleD

NA

Fig. 9.2 Schematic representation of a typical yeast episomalplasmid (YEp 24). The plasmid can replicate both in E. coli(due to the presence of the pBR322 origin of replication) andin S. cerevisiae (due to the presence of the yeast 2 µm origin ofreplication). The ampicillin and tetracycline determinants are derived from pBR322 and the URA3 gene from yeast.

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Cloning in S. cerevisiae and other fungi 159

Although plasmids containing an ars transformyeast very efficiently, the resulting transformantsare exceedingly unstable. For unknown reasons, YRpstend to remain associated with the mother cell andare not efficiently distributed to the daughter cell.(Note: S. cerevisiae does not undergo binary fissionbut buds off daughter cells instead.) Occasional stable transformants are found and these appear tobe cases in which the entire YRp has integrated intoa homologous region on a chromosome in a manneridentical to that of YIps (Stinchcomb et al. 1979,Nasmyth & Reed 1980).

Yeast centromere plasmids

Using a YRp vector, Clarke and Carbon (1980) isolated a number of hybrid plasmids containingDNA segments from around the centromere-linkedleu2, cdc10 and pgk loci on chromosome III of yeast.As expected for plasmids carrying an ars, most of therecombinants were unstable in yeast. However, oneof them was maintained stably through mitosis and meiosis. The stability segment was confined to a1.6 kb region lying between the leu2 and cdc10 lociand its presence on plasmids carrying either of twoars tested resulted in those plasmids behaving likeminichromosomes (Clarke & Carbon 1980, Hsiao &Carbon 1981). Genetic markers on the minichromo-somes acted as linked markers segregating in thefirst meiotic division as centromere-linked genes andwere unlinked to genes on other chromosomes.

Structurally, plasmid-borne centromere sequenceshave the same distinctive chromatin structure thatoccurs in the centromere region of yeast chromo-somes (Bloom & Carbon 1982). Functionally YCpsexhibit three characteristics of chromosomes inyeast cells. First, they are mitotically stable in theabsence of selective pressure. Secondly, they segreg-ate during meiosis in a Mendelian manner. Finally,they are found at low copy number in the host cell.

Yeast artificial chromosomes

All three autonomous plasmid vectors described aboveare maintained in yeast as circular DNA molecules – even the YCp vectors, which possess yeast cen-tromeres. Thus, none of these vectors resembles thenormal yeast chromosomes which have a linear

structure. The ends of all yeast chromosomes, likethose of all other linear eukaryotic chromosomes,have unique structures that are called telomeres.Telomere structure has evolved as a device to pre-serve the integrity of the ends of DNA molecules,which often cannot be finished by the conventionalmechanisms of DNA replication (for detailed dis-cussion see Watson 1972). Szostak and Blackburn(1982) developed the first vector which could bemaintained as a linear molecule, thereby mimickinga chromosome, by cloning yeast telomeres into aYRp. Such vectors are known as yeast artificial chro-mosomes (YACs).

One advantage of YACs is that, unlike the otherplasmid vectors, their stability increases as the size of the insert increases. Thus, there is no prac-tical limitation to the size of a YAC and they areessential tools in any genome-sequencing project.The method for cloning large DNA sequences inYACs developed by Burke et al. (1987) is shown inFig. 9.3.

Retrovirus-like vectors

The genome of S. cerevisiae contains 30–40 copies of a 5.9 kb mobile genetic element called Ty (forreview see Fulton et al. 1987). This transposable element shares many structural and functional features with retroviruses (see p. 193) and the copia element of Drosophila. Ty consists of a centralregion containing two long open reading frames(ORFs) flanked by two identical terminal 334 bprepeats called delta (Fig. 9.4). Each delta elementcontains a promoter as well as sequences recognizedby the transposing enzyme. New copies of the trans-poson arise by a replicative process, in which the Tytranscript is converted to a progeny DNA moleculeby a Ty-encoded reverse transcriptase. The comple-mentary DNA can transpose to many sites in thehost DNA.

The Ty element has been modified in vitro byreplacing its delta promoter sequence with pro-moters derived from the phosphoglycerate kinase or galactose-utilization genes (Garfinkel et al. 1985,Mellor et al. 1985). When such constraints are intro-duced into yeast on high-copy-number vectors, theTy element is overexpressed. This results in the formation of large numbers of virus-like particles

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(VLPs), which accumulate in the cytoplasm (Fig. 9.5).The particles, which have a diameter of 60–80 nm,have reverse-transcriptase activity. The major struc-tural components of VLPs are proteins produced by proteolysis of the primary translation product of

ORF 1. Adams et al. (1987) have shown that fusionproteins can be produced in cells by inserting part ofa gene from human immunodeficiency virus (HIV)into ORF 1. Such fusion proteins formed hybridHIV:Ty-VLPs.

The Ty element can also be subjugated as a vectorfor transposing genes to new sites in the genome.The gene to be transposed is placed between the 3′end of ORF 2 and the 3′ delta sequence (Fig. 9.6).Providing the inserted gene lacks transcription-termination signals, transcription of the 3′ deltasequence will occur, which is a prerequisite fortransposition. Such constructs act as amplificationcassettes, for, once introduced into yeast, transposi-tion of the new gene occurs to multiple sites in thegenome (Boeke et al. 1988, Jacobs et al. 1988).

Ligate

AMP

TRP1

pYAC vector

EcoRIcloning site

CEN4

ARS1

URA 3

TELTEL

ori

BamHI BamHI

BamHI andEcoRI-digest

TRP CENARS

URA

PartialEcoRI-digest

URA TELTRP CENARSTEL

Target DNA

Fig. 9.3 Construction of a yeastartificial chromosome containing largepieces of cloned DNA. Key regions of thepYAC vector are as follows: TEL, yeasttelomeres; ARS 1, autonomouslyreplicating sequence; CEN 4,centromere from chromosome 4; URA3and TRP1, yeast marker genes; Amp,ampicillin-resistance determinant ofpBR322; ori, origin of replication ofpBR322.

LTR LTR

Primary transcript

ORF 1

ORF 2

Fig. 9.4 Structure of a typical Ty element. ORF 1 and ORF 2represent the two open reading frames. The delta sequencesare indicated by LTR (long terminal repeats).

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Cloning in S. cerevisiae and other fungi 161

Choice of vector for cloning

There are three reasons for cloning genes in yeast.The first of these relates to the potential use of yeastas a cloning host for the overproduction of proteins ofcommercial value. Yeast offers a number of advant-ages, such as the ability to glycosylate proteins dur-ing secretion and the absence of pyrogenic toxins.Commercial production demands overproduction and

the factors affecting expression of genes in yeast arediscussed in a later section (see p. 165). Yeast is alsoused in the production of food and beverages. Theability to clone in yeast without the introduction ofbacterial sequences by using vectors like those of Chin-ery and Hinchliffe (1989) is particularly beneficial.

A second reason for cloning genes in yeast is theability to clone large pieces of DNA. Although thereis no theoretical limit to the size of DNA which can be cloned in a bacterial plasmid, large recombinantplasmids exhibit structural and segregative instabil-ity. In the case of bacteriophage-λ vectors, the size ofthe insert is governed by packaging constraints. ManyDNA sequences of interest are much larger than this.For example, the gene for blood Factor VIII coversabout 190 kbp, or about 0.1% of the human X chro-mosome, and the Duchenne muscular dystrophy genespans more than a megabase. Long sequences ofcloned DNA have greatly facilitated efforts to sequencethe human genome. YACs offer a convenient way to clone large DNA fragments but are being replacedwith BACs and PACs (see p. 67) because these havegreater stability. Nevertheless, the availability ofYACs with large inserts was an essential prerequisitefor the early genome-sequencing projects and theywere used in the sequencing of the entire S. cerevisiaechromosome III (Oliver et al. 1992). The method for

Fig. 9.5 Ty virus-like particles (magnification 80 000) carrying the entire HIV1 TAT coding region. (Photograph courtesy of Dr S. Kingsman.)

2µ ORI

Transcriptof geneinsert

P

δ

Ty transcript

pGALδ

URA 3

Fig. 9.6 Structure of the multicopy plasmid used for insertinga modified Ty element, carrying a cloned gene, into the yeastchromosome. pGAL and P are yeast promoters, δ representsthe long terminal repeats (delta sequences) and the red regionrepresents the cloned gene. (See text for details.)

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162 CHAPTER 9

Table 9.1 Properties of the different yeast vectors.

Vector

YIp

YEp

YRp

YCp

YAC

Ty

Transformationfrequency

102 transformantsper mg DNA

103–105

transformants per mg DNA

104 transformantsper mg DNA

104 transformantsper mg DNA

Depends onvector used tointroduce Tyinto cell

Copyno./cell

1

25–200

1–20

1–2

1–2

~20

Loss in non-selective

medium

Much less than1% pergeneration

1% pergeneration

Much greaterthan 1% pergeneration butcan getchromosomalintegration

Less than 1%per generation

Depends onlength: thelonger the YACthe morestable it is

Stable, sinceintegrated intochromosome

Advantages

1 Of all vectors, this kind give moststable maintenance of cloned genes2 An integrated YIp plasmid behavesas an ordinary genetic marker, e.g. a diploid heterozygous for anintegrated plasmid segregates theplasmid in a Mendelian fashion3 Most useful for surrogate geneticsof yeast, e.g. can be used to introduce deletions, inversions andtranspositions (see Botstein & Davis 1982)

1 Readily recovered from yeast2 High copy number3 High transformation frequency4 Very useful for complementationstudies

1 Readily recovered from yeast2 High copy number. Note that thecopy number is usually less than thatof YEp vectors but this may be usefulif cloning gene whose product isdeleterious to the cell if produced inexcess3 High transformation frequency4 Very useful for complementationstudies5 Can integrate into the chromosome

1 Low copy number is useful ifproduct of cloned gene is deleteriousto cell2 High transformation frequency3 Very useful for complementationstudies4 At meiosis generally showsMendelian segregation

1 High-capacity cloning systempermitting DNA molecules greaterthan 40 kb to be cloned2 Can amplify large DNA moleculesin a simple genetic background

Can get amplification followingchromosomal integration

Disadvantages

1 Low transformationfrequency2 Can only be recoveredfrom yeast by cuttingchromosomal DNA withrestriction endonucleasewhich does not cleaveoriginal vectorcontaining cloned gene

Novel recombinantsgenerated in vivo byrecombination withendogenous 2 mmplasmid

Instability oftransformants

Low copy number makesrecovery from yeastmore difficult than thatwith YEp or YRp vectors

Difficult to map bystandard techniques

Needs to be introducedinto cell in anothervector

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Cloning in S. cerevisiae and other fungi 163

cloning large DNA sequences developed by Burke etal. (1987) is shown in Fig. 9.3).

For many biologists the primary purpose of clon-ing is to understand what particular genes do in vivo.Thus most of the applications of yeast vectors havebeen in the surrogate genetics of yeast. One advant-age of cloned genes is that they can be analysed easily,particularly with the advent of DNA-sequencingmethods. Thus nucleotide-sequencing analysis canreveal many of the elements that control expressionof a gene, as well as identifying the sequence of the gene product. In the case of the yeast actin gene(Gallwitz & Sures 1980, Ng & Abelson 1980) andsome yeast transfer RNA (tRNA) genes (Peebles et al.1979, Olson 1981), this kind of analysis revealed thepresence within these genes of non-coding sequenceswhich are copied into primary transcripts. Theseintrons are subsequently eliminated by a processknown as splicing. Nucleotide-sequence analysis canalso reveal the direction of transcription of a gene,although this can be determined in vivo by othermethods. For example, if the yeast gene is expressedin E. coli using bacterial transcription signals, thedirection of reading can be deduced by observing theorientation of a cloned fragment required to permitexpression. Finally, if a single transcribed yeast geneis present on a vector, the chimera can be used as aprobe for quantitative solution hybridization analysisof transcription of the gene.

The availability of different kinds of vectors withdifferent properties (see Table 9.1) enables yeastgeneticists to perform manipulations in yeast likethose long available to E. coli geneticists with theirsex factors and transducing phages. Thus clonedgenes can be used in conventional genetic analysisby means of recombination using YIp vectors or linearized YRp vectors (Orr-Weaver et al. 1981).Complementation can be carried out using YEp,YRp, YCp or YAC vectors, but there are a number offactors which make YCps the vectors of choice (Roseet al. 1987). For example, YEps and YRps exist athigh copy number in yeast and this can prevent theisolation of genes whose products are toxic whenoverexpressed, e.g. the genes for actin and tubulin.In other cases, the overexpression of genes otherthan the gene of interest can suppress the mutationused for selection (Kuo & Campbell 1983). All theyeast vectors can be used to create partial diploids or

partial polyploids and the extra gene sequences canbe integrated or extrachromosomal. Deletions, pointmutations and frame-shift mutations can be intro-duced in vitro into cloned genes and the altered genesreturned to yeast and used to replace the wild-typeallele. Excellent reviews of these techniques havebeen presented by Botstein and Davis (1982), Hickset al. (1982), Struhl (1983) and Stearns et al. (1990).

Plasmid construction by homologousrecombination in yeast

During the process of analysing a particular clonedgene it is often necessary to change the plasmid’sselective marker. Alternatively, it may be desired to move the cloned gene to a different plasmid, e.g.from a YCp to a YEp. Again, genetic analysis mayrequire many different alleles of a cloned gene to beintroduced to a particular plasmid for subsequentfunctional studies. All these objectives can be ach-ieved by standard in vitro techniques, but Ma et al.(1987) have shown that methods based on recom-bination in vivo are much quicker. The underlying principle is that linearized plasmids are efficientlyrepaired during yeast transformation by recombina-tion with a homologous DNA restriction fragment.

Suppose we wish to move the HIS3 gene frompBR328, which cannot replicate in yeast, to YEp420(see Fig. 9.7). Plasmid pRB328 is cut with PvuI and PvuII and the HIS3 fragment selected. The HIS3fragment is mixed with YEp420 which has been linearized with EcoRI and the mixture transformedinto yeast. Two crossover events occurring betweenhomologous regions flanking the EcoRI site of YEp420will result in the generation of a recombinant YEpcontaining both the HIS3 and URA3 genes. TheHIS3 gene can be selected directly. If this were notpossible, selection could be made for the URA3 gene,for a very high proportion of the clones will alsocarry the HIS3 gene.

Many other variations of the above method havebeen described by Ma et al. (1987), to whom theinterested reader is referred for details.

Expression of cloned genes

When gene manipulation in fungi first became possible, there were many unsuccessful attempts to

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express heterologous genes from bacteria or highereukaryotes. This suggested that fungal promotershave a unique structure, a feature first shown for S. cerevisiae (Guarente 1987). Four structural elementscan be recognized in the average yeast promoter

(Fig. 9.8). First, several consensus sequences arefound at the transcription-initiation site. Two of thesesequences, TC(G/A)A and PuPuPyPuPu, accountfor more than half of the known yeast initiation sites (Hahn et al. 1985, Rudolph & Hinnen 1987).These sequences are not found at transcription-initiation sites in higher eukaryotes, which implies a mechanistic difference in their transcriptionmachinery compared with yeast.

The second motif in the yeast promoter is theTATA box (Dobson et al. 1982). This is an AT-richregion with the canonical sequence TATAT/AAT/A,located 60–120 nucleotides before the initiationsite. Functionally, it can be considered equivalent tothe Pribnow box of E. coli promoters (see p. 51).

The third and fourth structural elements areupstream activating sequences (UASs) and upstreamrepressing sequences (URSs). These are found ingenes whose transcription is regulated. Binding ofpositive-control proteins to UASs increases the rateof transcription and deletion of the UASs abolishestranscription. An important structural feature ofUASs is the presence of one or more regions of dyad symmetry (Rudolph & Hinnen 1987). Bindingof negative-control proteins to URSs reduces thetranscription rate of those genes that need to be negatively regulated.

The level of transcription can be affected bysequences located within the gene itself and whichare referred to as downstream activating sequences(DASs). Chen et al. (1984) noted that, when usingthe phosphoglycerate kinase (PGK ) promoter, sev-eral heterologous proteins accumulate to 1–2% of total cell protein, whereas phosphoglycerate kinaseitself accumulates to over 50%. These disappointingamounts of heterologous protein reflect the levels of

Recombination

HIS 3

pBR328

AmpR

SalBam

PvuIIPvuI

Eco

2µ

URA

3

HIS 3

Eco-cutYEp420

SalBam

PvuI

PvuI Sal

PvuII

Bam

Eco

PvuI + PvuII

Recombinant

2µURA

3

HIS

3

AmpR

EcoPvuI

BamSal

Fig. 9.7 Plasmid construction by homologous recombinationin yeast. pRB328 is digested with PvuI and PvuII and theHIS3-containing fragment is transformed into yeast alongwith the EcoRI-cut YEp420. Homologous recombinationoccurs between pBR322 sequences, shown as thin lines, togenerate a new plasmid carrying both HIS3 and URA3.

mRNA

UAS URS

InitiatorCoding

sequenceTATA

20–40 bp

40–120 bp

100–1400 bp

Fig. 9.8 Structure of typical yeast promoters. (See text for details.)

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Cloning in S. cerevisiae and other fungi 165

mRNA which were due to a lower level of initiationrather than a reduced mRNA half-life (Mellor et al.1987). Addition of downstream PGK sequencesrestored the rate of mRNA transcription, indicatingthe presence of a DAS. Evidence for these DASs hasbeen found in a number of other genes.

Overexpression of proteins in fungi

The first overexpression systems developed were for S. cerevisiae and used promoters from genes

encoding abundant glycolytic enzymes, e.g. alcoholdehydrogenase (ADH1), PGK or glyceraldehyde-3-phosphate dehydrogenase (GAP). These are strongpromoters and mRNA transcribed from them canaccumulate up to 5% of total. They were at firstthought to be constitutive but later were shown tobe induced by glucose (Tuite et al. 1982). Now thereis a large variety of native and engineered promotersavailable (Table 9.2), differing in strength, regulationand induction ratio. These have been reviewed indetail by Romanos et al. (1992).

Table 9.2 Common fungal promoters used for manipulation of gene expression.

Species Promoter Gene Regulation

GeneralS. cerevisiae PGK Phosphoglycerate kinase Glucose-induced

GAL1 Galactokinase Galactose-inducedPHOS Acid phosphatase Phosphate-repressedADH2 Alcohol dehydrogenase II Glucose-repressedCUP1 Copper metallothionein Copper-inducedMFa1 Mating factor a1 Constitutive but temperature-induced

variant availableCandida albicans MET3 ATP sulphur lyase Repressed by methionine and cysteine

Methanol utilizersCandida boidnii AOD1 Alcohol oxidase Methanol-inducedHansenula polymorpha MOX Alcohol oxidase Methanol-inducedPichia methanolica AUG1 Alcohol oxidase Methanol-inducedPichia pastoris AOX1 Alcohol oxidase Methanol-induced

GAP Glyceraldehyde-3-phosphate Strong constitutivedehydrogenase

FLD1 Formaldehyde dehydrogenase Methanol- or methylamine-inducedPEX8 Peroxin Methanol-inducedYPT1 Secretory GTPase Medium constitutive

Lactose utilizerKluyveromyces lactis LAC4 b-Galactosidase Lactose-induced

PGK Phosphoglycerate kinase Strong constitutiveADH4 Alcohol dehydrogenase Ethanol-induced

Starch utilizerSchwanniomyces AMY1 a-Amylase Maltose- or starch-inducedoccidentalis

GAM1 Glucoamylase Maltose- or starch-induced

Xylose utilizerPichia stipitis XYL1 Xylose-induced

Alkane utilizerYarrowia lipolytica XPR2 Extracellular protease Peptone-induced

TEF Translation elongation factor Strong constitutiveRPS7 Ribosomal protein S7 Strong constitutive

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The ideal promoter is one that is tightly regulatedso that the growth phase can be separated from the induction phase. This minimizes the selection ofnon-expressing cells and can permit the expressionof proteins normally toxic to the cell. The ideal promoter will also have a high induction ratio. Onepromoter which has these characteristics and whichis now the most widely used is that from the GAL1gene. Galactose regulation in yeast is now extremelywell studied and has become a model system foreukaryotic transcriptional regulation (see Box 9.1).

Following addition of galactose, GAL1 mRNA israpidly induced over 1000-fold and can reach 1% of total mRNA. However, the promoter is stronglyrepressed by glucose and so in glucose-grown cul-tures this induction only occurs following depletionof glucose. To facilitate galactose induction in thepresence of glucose, mutants have been isolated whichare insensitive to glucose repression (Matsumoto et al. 1983, Horland et al. 1989). The trans-activatorGAL4 protein is present in only one or two moleculesper cell and so GAL1 transcription is limited. Withmulticopy expression vectors, GAL4 limitation isexacerbated. However, GAL4 expression can be madeautocatalytic by fusing the GAL4 gene to a GAL10promoter (Schultz et al. 1987), i.e. GAL4 expressionis now regulated (induced) by galactose.

In recent years, methylotrophic yeasts, such asPichia pastoris, have proved extremely popular ashosts for the overexpression of heterologous pro-teins. There are a number of reasons for this. First,the alcohol oxidase (AOX1) promoter is one of thestrongest and most regulatable promoters known.Second, it is possible to stably integrate expressionplasmids at specific sites in the genome in either single or multiple copies. Third, the strains can becultivated to very high density. To date, over 300foreign proteins have been produced in P. pastoris(Cereghino & Cregg 1999, 2000). Promoters for usein other yeasts are shown in Table 9.2.

Specialist vectors

Many different specialist yeast vectors have beendeveloped which incorporate the useful featuresfound in the corresponding E. coli vectors (see p. 70), e.g. an f1 origin to permit sequencing ofinserts production of the cloned gene product as a

purification fusion, etc. Some representative exam-ples are shown in Fig. 9.9. Two aspects of these vectors warrant further discussion: secretion andsurface display.

In yeast, proteins destined for the cell surface orfor export from the cell are synthesized on andtranslocated into the endoplasmic reticulum. Fromthere they are transported to the Golgi body for processing and packaging into secretory vesicles.Fusion of the secretory vesicles with the plasmamembrane then occurs constitutively or in responseto an external signal (reviewed by Rothman & Orci1992). Of the proteins naturally synthesized andsecreted by yeast, only a few end up in the growthmedium, e.g. the mating pheromone α factor andthe killer toxin. The remainder, such as invertaseand acid phosphatase, cross the plasma membranebut remain within the periplasmic space or becomeassociated with the cell wall.

Polypeptides destined for secretion have ahydrophobic amino-terminal extension, which isresponsible for translocation to the endoplasmicreticulum (Blobel & Dobberstein 1975). The exten-sion is usually composed of about 20 amino acidsand is cleaved from the mature protein within theendoplasmic reticulum. Such signal sequences pre-cede the mature yeast invertase and acid phos-phatase sequences. Rather longer leader sequencesprecede the mature forms of the α mating factor and the killer toxin (Kurjan & Herskowitz 1982,Bostian et al. 1984). The initial 20 amino acids or soare similar to the conventional hydrophobic signalsequences, but cleavage does not occur in the endo-plasmic reticulum. In the case of α factor, which hasan 89 amino acid leader sequence, the first cleavageoccurs after a Lys–Arg sequence at positions 84 and85 and happens in the Golgi body ( Julius et al. 1983,1984).

To date, a large number of non-yeast polypeptideshave been secreted from yeast cells containing theappropriate recombinant plasmid and in almost allcases the α-factor signal sequence has been used.There is a perception that S. cerevisiae has a lowersecretory capacity than P. pastoris and other yeasts(Muller et al. 1998), but the real issue may be the type of vector used. For example, Parekh et al.(1996) found that S. cerevisiae strains containingone stably integrated copy of an expression cassette

POGC09 9/11/2001 11:05 AM Page 166

Galactose is metabolized to glucose-6-phosphate inyeast by an identical pathway to that operating inother organisms (Fig. B9.1). The key anzymes andtheir corresponding genes are a kinase (GAL1), atransferase (GAL7), an epimerase (GAL10) and amutase (GAL5). Melibiose (galactosyl-glucose) ismetabolized by the same enzymes after cleavage by an α-galactosidase encoded by the MEL1 gene.Galactose uptake by yeast cells is via a permeaseencoded by the GAL2 gene. The GAL5 gene isconstitutively expressed. All the others are induced by growth on galactose and repressed during growthon glucose.

The GAL1, GAL7 and GAL10 genes are clusteredon chromosome II but transcribed separately fromindividual promoters. The GAL2 and MEL1 genes areon other chromosomes. The GAL4 gene encodes a

protein that activates transcription of the catabolicgenes by binding UAS 5′ to each gene. The GAL80gene encodes a repressor that binds directly to GAL4gene product, thus preventing it from activatingtranscription. The GAL3 gene product catalyses the conversion of galactose to an inducer, whichcombines with the GAL80 gene product, preventing it from inhibiting the GAL4 protein from binding toDNA (Fig. B9.2).

The expression of the GAL genes is repressedduring growth on glucose. The regulatory circuitresponsible for this phenomenon, termed cataboliterepression, is superimposed upon the circuitresponsible for induction of GAL gene expression.Very little is known about its mechanism.

For a review of galactose metabolism in S. cerevisiae,the reader should consult Johnston (1987).

Box 9.1 Galactose metabolism and its control in Saccharomyces cerevisiae

GalactoseMelibiose Galactose Gal–1–P Glu–1–P Glu–6–P Glycolysis

MEL1α-galactosidase

GAL2Permease

GAL1Kinase

GAL7Transferase

GAL5Mutase

Medium Cytoplasm

UDP–Glu UDP–Gal

EpimeraseGAL10

GAL3

GAL80 protein complexesGAL4 protein and preventsactivation of GAL transcription

GAL80protein

Galactose Inducer

GAL4 protein binds to UASsand activates GAL transcription

GAL80 protein–inducercomplex

Noinducer

Fig. B9.1 The genes and enzymes associated with the metabolism of galactose by yeast.

Fig. B9.2 The regulation of transcription of the yeast galactose genes.

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168 CHAPTER 9

secreted more bovine pancreatic trypsin inhibitorthan strains with the same expression cassette on a2 µm multicopy vector. Optimal expression wasobtained with 10 integrated copies. With those proteins which tend to accumulate in the endoplas-mic reticulum as denatured aggregates, secretionmay be enhanced by simultaneously overexpressingchaperons (Shusta et al. 1998). Secretion may alsobe enhanced by minor amino acid changes. Katakuraet al. (1999) noted a sixfold increase in lactoglobulinsecretion by conversion of a tryptophan residue totyrosine.

Yeast surface display

S. cerevisiae can be used to elucidate and dissect thefunction of a protein in a manner similar to phage-display systems. Either can be used to detect protein–

ligand interactions and to select mutant proteinswith altered binding capacity (Shusta et al. 1999).However, phage-display systems often cannot dis-play secreted eukaryotic proteins in their nativefunctional conformation, whereas yeast surface display can.

Yeast surface display makes use of the cell surfacereceptor α-agglutinin (Aga), which is a two-subunitglycoprotein. The 725-residue Aga1p subunit anchorsthe assembly to the cell wall via a covalent linkage.The 69-residue binding subunit (Aga2p) is linked toAga1p by two disulphide bonds. To achieve surfacedisplay, the appropriate gene is inserted at the C terminus of a vector-borne Aga2p gene under thecontrol of the GAL1 promoter. The construct is thentransformed into a yeast strain carrying a chro-mosomal copy of the Aga1p gene, also under thecontrol of the GAL1 promoter. If the cloned gene has

Pml I

CYC1 pA

2µorigin

f1or

i

PGAL 1

pUCori

Am

plicillin

Blasticidin

CEN

6/ARSH4

URA 3

TRP1

YESVectors

Hin

d II

IAs

p718

IKp

n I

Sac

IBa

mH

IBs

tX I

EcoR

IBs

tX I

Not

IX

ho I

Xba

I*

Polylinkers for C-terminal tagged vectors

V5 epitope 6xHis STOP

Pme

I

Hin

d II

I Asp7

18 I

Kpn

IBa

mH

IBs

tX I

EcoR

IBs

tX I

Not

IX

ho I

Xba

I

Polylinkers for N-terminal tagged vectors

ATG 6xHis STOP

Pme

I

Xpress Epitope EK Site V5 epitope 6xHis

5’AOX1

AOX1 TT BamH I

PTEF1

PE M

7Z

eocin

CYC1 TT

ColE1

pPICZ A, B, Cand

pPICZα A, B, C3.3 kb

Bgl II

BstB

IEc

oR I

Pml I

Sfi I

Bsm

B I

Asp7

18 I

Kpn

IX

ho I

Sac

IIN

ot I

Apa

I*

pPICZ A, B, C

c-myc epitope 6xHis STOP

Xba

I*

SnaB

I*

pPICZα A, B, C

Pst

I*Ec

oR I

Sfi I

Bsm

B I

Asp7

18 I

Kpn

IX

ho I

Sac

IIN

ot I c-myc epitope 6xHis STOP

Xba

I

Cla

I*α-factor

T7

T7

Fig. 9.9 Two specialized vectors for use in Saccharomyces (YES vectors) and Pichia (pPICZ). V5, Express, 6XHis and c-myc encodeepitopes which can be readily detected and purified by affinity chromatography. The YES vectors offer a choice of 2 µm origin forhigh copy or CEN6/ARSH4 origin for low copy in yeast, a choice of URA3 or TRP1 genes for auxotrophic selection on minimalmedium, blasticidin resistance for dominant selection in any strain and an f1 origin to facilitate DNA sequencing. In the pPICZvectors, the zeocin-resistance gene is driven by the EM-7 promoter for selection in E. coli and the TEF1 promoter for selection inPichia. More details of these features can be found in Chapter 5. (Figure reproduced courtesy of Invitrogen Corporation.)

POGC09 9/11/2001 11:05 AM Page 168

Cloning in S. cerevisiae and other fungi 169

been inserted in the correct translational readingframe, its gene product will be synthesized as afusion with the Aga2p subunit. The fusion productwill associate with the Aga1p subunit within thesecretory pathway and be exported to the cell sur-face (Boder & Wittrup 1997).

In practice, the gene fusion is somewhat morecomplicated. Usually the cloned gene product issandwiched between two simple epitopes to permitquantitation of the number of fusion proteins per cell and to determine the accessibility of differentdomains of the fusion protein (Fig. 9.10).

Detecting protein–protein interactions

Chien et al. (1991) have made use of the properties ofthe GAL4 protein to develop a method for detectingprotein–protein interactions. The GAL4 protein hasseparate domains for the binding to UAS DNA andfor transcriptional activation. Plasmids were con-structed which encode two hybrid proteins. The firstconsisted of the DNA-binding domain (residues1–147) of the GAL4 protein fused to a test protein.The second consisted of residues 768–881 of theGAL4 protein, representing the activation domain,fused to protein sequences encoded by a library ofyeast genomic DNA fragments. Interaction betweenthe test protein (bait) and a protein encoded by one of the library plasmids (prey) led to transcriptionalactivation of a reporter gene (Fig. 9.11). This methodis known as the two-hybrid system. The two-hybridsystem has become a major tool in the study of protein–protein and protein–ligand interactions (see

c-myc

Clonedgene

product

HA

Aga 2

s

s

s

s

Aga 1

Aga2 cassette

TRP

TRP 1

CEN

/ARS

ori

P GA1

AmpicillinR

Aga 2 HA MCS c-myc

Yeast cell wall

(a) (b)

Fig. 9.10 Yeast surface display ofheterologous proteins. (a) Schematicrepresentation of surface display. (b) A vector used for facilitating surfacedisplay. MCS, multiple cloning site; HA, haemagglutinin epitope; c-myc, c-myc epitope. See text for details.

DNA-binding domain hybrid

BAIT

DBD GAL4 (1–147)

GAL1–lacZUASG

Y1-n

Activation domain hybrids encoded by a library

GAL1–lacZUASG

TAD

GAL4 (768–881)

Interaction between DNA-binding domainhybrid and a hybrid from the library

BAIT

DBD GAL4 (1–147)

GAL1–lacZUASG

Yi

TAD

GAL4 (768–881)PREY

PREY

Fig. 9.11 Strategy to detect interacting proteins using the two-hybrid system. UASG is the upstream activatingsequence for the yeast GAL genes, which binds the GAL4protein, DBD is a DNA-binding domain and TAD is atranscription-activation domain. Interaction is detected byexpression of a GAL1– lacZ gene fusion. (Reproduced courtesyof Dr S. Fields and the National Academy of Sciences.)

POGC09 9/11/2001 11:05 AM Page 169

Post-translational modifications

A limitation of using the two-hybrid system is thatcertain protein–protein interactions are not possiblein S. cerevisiae because of the absence of theappropriate post-translational modifying enzymes.This is a particular problem with baits relevant tosignal transduction in higher eukaryotes, because of the prevalence of site-specific phosphorylation by tyrosine kinase as a means of regulating protein–protein interactions. Osborne et al. (1996) were ableto solve this problem by using a strain of yeastexpressing the mammalian tyrosine kinase Lck.

Protein–peptide interactions

The utility of the two-hybrid system can be extendedto interactions between proteins and very small (< 16 residues) peptides (Yang et al. 1995, Colas et al. 1996). Small peptides can be used not only as the prey, as in the studies just cited, but also as the bait (Geyer et al. 1999, Norman et al. 1999). In another variation, Stagljar et al. (1996) havedescribed a rapid method to identify smallinteraction-specific sequences within larger proteins.In their method, complementary DNA (cDNA)encoding a known interacting protein is sheared by sonication and shotgun-cloned into a prey vector. The resulting targeted library is cotransformed with the bait of interest and direct selection made for fragments capable of conferring the desiredinteraction.

Three-component systems

In some cases, a bait and prey may not directlyinteract, or else form a transient contact, because athird component is missing. However, co-expression

of the bait with a previously identified partner protein may permit expression of the reporter gene (Tomashek et al. 1996). The ternary partner in such three-component interactions need not be a protein but can be a small molecule, such as a hormone (Lee et al. 1995) or a drug (Chiu et al. 1994).

Bait and hook

The two-hybrid system can be modified to study the interaction between proteins and RNA (SenGupta et al. 1999) or proteins and drugs (Licitra & Liu 1996). To do this a ‘hook’ is used todisplay an RNA molecule or a drug to the incomingprey, thereby forming a non-protein bridge to thereporter gene (Fig. B9.3).

Reverse two-hybrid system

The original two-hybrid system relies ontranscriptional activation of reporter genes to detect protein–protein interactions (Fig. 9.11).However, the two-hybrid system can be ‘turned on itshead’ to monitor loss of protein–protein interactions.For example, Vidal et al. (1996a,b), have described ascreen for mutations that disrupted the ability of thetranscription factor E2F1 to associate with its partnerDP1. In the absence of interaction between the twopartner proteins, transcription of the URA3 reportergene was abolished. This in turn allowed the hostyeasts to grow in the presence of 5-fluoro-orotic acid, a compound that is toxic to cells capable ofsynthesizing uracil. This type of reverse selection can be used to screen for peptides or other smallmolecules that disrupt protein–protein interactionsand that could have utility as drugs (Huang &Schreiber 1997).

Box 9.2 Variations of the two-hybrid system

UAS

DBD

Hook

Prey TAD

Reporter

Bait RNA

Transcription

Fig. B9.3 The bait-and-hookvariation of the two-hybrid system.

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Cloning in S. cerevisiae and other fungi 171

Box 9.2 and the reviews by Colas & Brent 1998, andUetz & Hughes 2000).

A major operational problem with the two-hybridsystem has been the high frequency of false positives,i.e. cells in which the reporter gene is active eventhough the bait and prey do not interact in natureand/or in yeast. There are two general solutions tothis problem. In the first of these ( James et al. 1996),a host strain is used that contains three reportergenes under the control of different GAL4-induciblepromoters. An alternative approach is to use thetwo-bait system (Fig. 9.12). In this system, the yeastexpresses a single prey and two independent baits,each targeted to distinct reporter genes (Xu et al.1997). In weeding out false positives, one looks forpreys that interact with only one bait. The rationalehere is that the more related the two baits are, themore likely it is that a prey interacting with only oneof them is a natural interaction.

Identifying genes encoding particularcellular activities

As a result of a major genome-sequencing project,the sequences of genes encoding every biochemicalactivity of S. cerevisiae are known. The next task is to connect specific biochemical activities with par-ticular genes. Martzen et al. (1999) have developed a rapid and sensitive method for doing this which is applicable to almost any activity. The starting-point was a yeast vector containing the glutathione-S-transferase (GST) gene under the control of theCUP1 promoter. A large number of different DNAfragments bearing yeast ORFs were cloned in thisvector to generate GST–ORF fusions. In all, 6144

constructs were made and these were transformedback into S. cerevisiae.

To correlate genes with activity, the different trans-formants were dispensed into 64 96-well microtitreplates (64 × 96 = 6144). Initially, all the clones fromeach plate were pooled and the 64 pools analysed fora specific biochemical activity. Analysis is facilitatedsince the GST–ORF fusions are readily purified fromother cellular activities by affinity chromatographyusing immobilized glutathione. Once a plate wasidentified as having the desired activity, pools ofclones were made from each of the eight rows and12 columns of wells and these 20 pools reassayed. Ifonly one clone expresses the desired activity, then onerow and one column should be identified (Fig. 9.13)and the point of intersection identifies the wantedclone. Sequencing of the ORF then allows identifica-tion of the gene corresponding to the activity.

Determining functions associated with particular genes

Determining the function of a particular gene productusually involves determining the expression profile ofthe gene, the subcellular location of the protein, andthe phenotype of a null strain lacking the protein.Conditional alleles of the gene are often created as an additional tool. These procedures usually requiremultiple independent manipulations. Ross-Macdonaldet al. (1997) have developed a multifunctional,transposon-based system that simultaneously gener-ates constructs for all of the above analyses and issuitable for mutagenesis of any given S. cerevisiaegene. The transposons used by them are shown inFig. 9.14. Each carries a reporter gene (β-galactosidase

Transcription

UAS1

DBD1

Bait1 PreyTAD

Reporter 1

UAS2

DBD2

Bait2

Prey TAD

Reporter 2

Fig. 9.12 The dual-bait two-hybridsystem. The first DNA-binding domainfusion protein (DBD1–Bait1) drives the expression of reporter 1 throughUAS1. A second separate DNA-bindingdomain fused to a distinct bait(DBD2–Bait2) directs expression ofreporter 2 through UAS2. In thisexample, the prey can interact with Bait 1 but not Bait 2.

POGC09 9/11/2001 11:05 AM Page 171

172 CHAPTER 9

or green fluorescent protein) lacking a promoter. In-frame fusions between a yeast coding region andthe transposon can be detected by β-galactosidaseactivity or fluorescence. The transposon insertion creates a truncation of the gene, thereby generatinga null phenotype. The method is also adaptable to genome-wide analyses, using a high-throughputscreening methodology (Ross-Macdonald et al. 1999).

To use this transposon system, yeast genes arecloned in an E. coli strain that overexpresses the Tn3transposase. The transposon, carried on a derivativeof the sex factor F, is introduced by mating and trans-position ensues. Yeast DNA containing the transposonis excised from the E. coli vector and transformed backinto yeast, with selection made for URA3 activity. Twotypes of transformants are obtained. If homologous

64 poolseach with96 GST–ORFfusions

1 positive pool

1 positive row and1 positive column

1 positive strain

Fig. 9.13 Method for selectingglutathione-S-transferase open-reading-frame fusions. (Figurereproduced from Trends in Geneticscourtesy of Elsevier Press.)

Fig. 9.14 Schematic representation ofthe transposons, and the derived HATtag elements, for use in yeast. Eachtransposon carries the tetracycline-resistance determinant, a functionalyeast URA3 gene, and the res elementfrom transposon Tn3. The function of the res element is to resolvetransposition cointegrates. (Figurereproduced courtesy of Dr MichaelSnyder and the National Academy ofSciences.)

mTn-4xHA/lacZ

TR loxRHA lacZ URA3 tet res 3xHa

loxP TR

+cre 262-bpHAT tag

mTn-3xHA/lacZ

Xa

+cre274-bpHAT tag

mTn-3xHA/GFPGFP

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Cloning in S. cerevisiae and other fungi 173

recombination occurs at the URA3 locus, then noreporter activity is seen. If integration occurs at thelocus of the gene carrying the transposon, reporteractivity is retained but gene function is lost (Fig. 9.14).This reporter activity can be used to study the expres-sion of the cloned gene under different growth condi-tions. If the transformants also carry a phage P1 cregene (see p. 68), under the control of the GAL1 pro-moter, excision of most of the transposon sequencescan be induced by growth of the host strain in thepresence of galactose. In most instances, this resultsin restoration of gene activity and the gene productcan be purified by affinity purification, using anti-bodies to the haemagglutinin determinants.

An alternative approach to the large-scale muta-tional analysis of the yeast genome is to individuallydelete each gene using a polymerase chain reaction(PCR)-based methodology (Winzeler et al. 1999).For each gene, a deletion cassette is constructed (Fig. 9.15) that contains a kanamycin resistancegene, two ‘molecular bar-codes’ and yeast sequenceshomologous to the upstream and downstream flank-ing sequences. This construct is amplified by PCR andtransformed into a diploid yeast strain, where the dele-tion cassette replaces one of the two chromosomalgene sequences. The diploid strains are then allowedto sporulate and those haploid segregants that areviable are recovered.

The molecular bar-codes are particularly importantsince they are unique to each deletion strain withina pool of many different strains (Shoemaker et al.1996). The bar-code can be amplified by PCR, usingcommon primer sequences that flank the bar-code.A pool of yeast mutant strains can thus be analysedin a single experiment by amplifying the bar-codesand probing a DNA microarray (p. 116) containingsequences complementary to the bar-codes. Variationson this technique have been used in other organismsto identify key functions associated with particularactivities. For example, signature-tagged mutagenesishas been used to identify genes essential for virulence(for review, see Lehoux & Levesque 2000).

The application of genome-wide mutagenesis tech-niques to the understanding of the ways in whichthe different components of the yeast cell interacthas been reviewed by Delneri et al. (2001) and Vidanand Snyder (2001).

Fig. 9.15 ‘Knockout’ strain collection. (a) The bar-codeddeletion cassette is generated in two rounds of PCR. Itcontains a kanamycin-resistance gene for selection in yeastand two molecular bar-codes 20 bp long that are unique toeach deletion. The molecular bar-codes are flanked bycommon sequence elements that allow for the PCRamplification of all the bar-codes in a population of yeaststrains. At the end of the cassette are 45 bp sequences that are homologous to sequences flanking the gene of interest. (b) The deletion cassette is transformed into diploid yeaststrains. The 45 bp of yeast sequences allows one copy of thegene of interest to be replaced with the deletion cassette viahomologous recombination. The resulting heterozygousstrains (c) can then be sporulated and dissected to generatea collection of haploid mutant strains, if viable (d).Homozygous mutant strains (e) are made by the selectivematings of haploid segregants. (Figure reproduced courtesy of Elsevier Science Ltd and the authors.)

(a) PCR deletion cassette

45 bp bc KanR bc 45 bp

(b) Homologous recombination

45 bp bc KanR bc 45 bp

(c) Heterozygous yeast strain

Yeast ORF

Yeast ORF

bc KanR bc

Yeast ORF

(d) Haploid yeast strain

bc KanR bc

(e) Homozygous diploid yeast strain

bc KanR bc

bc KanR bc

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