4Yeast Molecular TechniquesIn this overview, we will concentrate on approaches that havebeen useful in yeast recombinant DNA technology ratherthan consider the plethora of genetic and biochemical tech-niques that have made yeast biology so successful over thepast decades.

Some standard compilations of general proceduresemployed in studying structural, genetic or biochemicalaspects of yeast cells (Broach, Pringle, and Jones, 1991;Guthrie and Fink, 1991; Mortimer et al., 1992; Johnston,1994) have already been mentioned in the Introduction.

4.1Handling of Yeast Cells

4.1.1Growth of Yeast Cells

In the laboratory, yeast cells can be grown in liquid culture (forminipreparations or inmass cultures) or on agar plates, wherethey can be viewed as single colonies, when applied at appro-priate dilutions. Agar plates are advantageous for replica plat-ing and colony hybridization (Grunstein andHogness, 1975).

Depending on the conditions that have to be chosen forexperimental studies, there is a multitude of recipes how toprepare suitable liquid media. For many purposes, supplierskeep defined yeast media on stock. Relevant information isavailable from the corresponding catalogs or brochures.

At the beginning, it may be useful to briefly mentionsome of the approaches that are still in use to produce yeastcells synchronized in terms of cell cycle phase. Synchroniza-tion of cell growth (meaning that each single cell in a popula-tion has reached the same status of the cell cycle) can beachieved in two ways. (i) The cells are blocked in the S phaseor M phase by specific inhibitors, followed by continuous-flow centrifugation. A specialized device formerly used inthis procedure is the so-called elutriator. Beckman offered aspecial rotor system (Rotor-Beckman JEB6 Elutriator RotorAssembly for the Avanti J-Series of elutriator centrifuges).Elutriation can separate lighter particles from heavier ones(the latter in this case are the budding cells). (ii) A cell cycleblock is induced at the G1 phase by treatment of cells withmating pheromone, followed by gradient centrifugation,which is the preferred technique.

4.1.2Isolation of Particular Cell Types and Components

Before isolation procedures can be approached, the rigid cellwall of yeast cells has to be opened. There are various meth-ods to disrupt yeast cells, depending on if the cellular constit-uents have to be kept intact or will be of interest insubsequent isolation procedures.

A thorough breakage of cell walls is obtained by puttingfrozen yeast cells through a French Press or an Eaton (orHughes) press under high pressure. Both of these instru-ments consist of a thick metal (V4A steel) cylinder, about12 cm wide and 18 cm high, in whose central hole (diameterof about 3 cm) a rigid, tight metal stamp can be movedhydraulically. Before pressing, the hole is filled with a slurryof yeast cells cooled down in liquid nitrogen. The outlet atthe bottom of the cylinder is either a hole (about 2mm wide)for the French press or a narrow slit (about 1mm wide) forthe Eaton (or Hughes) press. The cell sap is collected and ismostly used for the preparation of yeast proteins or enzymes.

A more gentle procedure of opening yeast cells is usingglass bead homogenization in a vortex mixer or Braunhomogenizer. Also, repeated freezing in liquid nitrogen andthawing is a rather efficient, temperate disruption procedure.For the preparation of high-molecular-weight DNA, severalprocedures are in use, which rely on treatment with efficienttensides (see, e.g., Section 4.2.3).

The isolation of yeast spheroplasts, intact nuclei, respira-tory-competent mitochondria, and other subcellular compo-nents is summarized in Table 4.1.

4.2Genetic Engineering and Reverse Genetics

4.2.1Molecular Revolution

In the early 1970s, three events revolutionized molecularbiology: (i) the discovery of restriction mechanisms in bacte-rial cells by Werner Arber (Arber, 1965; Arber, 1978) onwhich basis the first specific restriction endonucleases couldbe isolated and applied by Smith and Wilcox (1969) and byAdler and Nathans (1973); (ii) the possibility of to create

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Yeast: Molecular and Cell Biology, Second Edition. Edited by Horst Feldmann.# 2012 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2012 by Wiley-VCH Verlag GmbH & Co. KGaA.

recombinant DNA in vitro and to transfer it into host cellswhere it is capable of exerting particular functions, first dem-onstrated by Paul Berg and collaborators (Jackson, Symons,and Berg, 1972; Berg, 1981); and (iii) the development ofmethods allowing the determination of DNA sequences byWalter Gilbert and coworkers (Maxam and Gilbert, 1977;Gilbert, 1981) and Frederick Sanger and collaborators (San-ger, Nicklen, and Coulson, 1977; Sanger, 2001), which had tofollow principles different from those applied to RNAsequencing. Not surprisingly, yeast molecular biology wassoon caught by these new potentials.

Until 1976 or 1977, any nucleic acid material from yeasthad to be isolated from cell preparations. Information onparticular genes and their regulation or interactions waslargely derived from genetic experiments – a privilege thatwas also offered by other organisms that had been used asgenetic model systems, such as bacteria and their phages,Neurospora, or Drosophila.

The beginning of genetic engineering undoubtedly wasmarked by the successful approach of Paul Berg and his col-laborators to show that recombinant DNA could be main-tained in a host cell (Jackson, Symons, and Berg, 1972). Ivividly remember a long night session with a full moon inthe courtyard of a monastery at a Summer School 1971 heldin Erice, where Berg, Sanger, and Tomkins chaired a discus-sion on the above three paradigm shifts. Restriction enzymesfrom a variety of sources soon became available and were

applied to generate recombinant DNA. Methods allowingthe determination of DNA sequences became a reality in theyears to follow and were used in yeast.

Although several methods had been developed for clon-ing and characterizing recombinant DNA molecules since1972 (Grunstein and Hogness, 1975), it was only after theAsilomar Conference on Recombinant DNA (Berg et al.,1975) that safe and simple procedures and vehicles couldbe propagated for extensive use in cloning recombinantDNA molecules. Refined cloning systems along theselines were developed in the years to follow. Clearly, theease of cloning was manifested by the use of plasmid vec-tors (Bolivar et al., 1977a; Bolivar et al., 1977b; Itakuraet al., 1977; Sutcliffe, 1978; Soberon, Covarrubias, andBolivar, 1980), but the big advantage of cloning vehiclesbased on phage l was the larger size of DNA sequencesthat could be accommodated (Blattner et al., 1977; Leder,Tiemeier, and Enquist, 1977). These properties wereshared by the cosmids – plasmid gene-cloning vectorspackageable in phage l heads (Collins and Hohn, 1978).A technical innovation – colony hybridization – alsobecame extremely useful in isolating specific genes fromyeast (Grunstein and Hogness, 1975).

Already before the safer cloning vehicles were available,plasmids and phage l had been used to clone gene-containing DNA fragments from a variety of organisms(Hollenberg, Kustermann-Kuhn, and Royer, 1976; Tiollais

Table 4.1 Isolation of subcellular entities.

Entity Procedure Selected references

Spheroplasts treatment of cells with enzymes digesting the cell wall: (i) glusulase orhelicase (snail gut juice), (ii) lyticase (Arthrobacter luteus), or (iii) zymolase(A. luteus)

Nuclei (intact) spheroplasts lysed with Ficoll in a homogenizer, differential centrifugationof lysate; further: Percoll or sorbitol gradient centrifugation

Gregory, Barbaric, and H€orz, 1998

DNA lysis of spheroplasts and centrifugation Wach et al., 1994b; Stucka and Feldmann,1994

rDNA 8–10% of total DNA is GC-rich and can be separated as a single peak(g-DNA) by cesium sulfate gradient centrifugation

tRNA þ 5S RNA extraction of suspended cells with cold phenol and ethanol precipitationfrom the aqueous phase

Monier, Stephenson, and Zamecnik, 1960

rRNA extraction of cells with hot phenol and ethanol precipitation from theaqueous phase

mRNA isolation and measurement of Mrna stability Piper, 1994; Brown, 1994Cell walls differential centrifugation and fractionation by chemical treatment Catley, 1988; Fleet, 1991Plasma membrane Panaretou and Piper, 1996Spindle pole bodies Rout and Kilmartin, 1994Nuclear envelopes Strambio-de-Castilla et al., 1995Vacuoles purification and in vitro analysis Cabrera and Ungermann, 2008Golgi membranes Lupashin, Hamamoto, and Schekman,

1996; Blanchette, Abazeed, and Fuller,2004

ER membranes Scott and Schekman, 2008Mitochondria(respirationcompetent)

gentle disruption of cells and centrifugation; supernatant centrifuged twicein sucrose gradients

Herrmann et al., 1994; Gregg, Kyryakov,and Titorenko, 2009

Peroxisomes Distel et al., 1996

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et al., 1976; Velten, Fukada, and Abelson, 1976), includingyeast (Hollenberg, 1982). For the first time, in vitro synthe-sized gene sequences were cloned, such as the b-globingene (Maniatis et al., 1976). As soon as the new cloningsystems appeared on the market, they were widely used toclone DNA fragments and particular genes from varioussources, including yeast (Rose and Broach, 1991).

4.2.2Transformation of Yeast Cells

The transformation of yeast cells by replicating hybrid plas-mids was independently developed by two laboratories asearly as 1978 – those of Jean Beggs (Beggs, 1978) and AlbertHinnen (Hinnen, Hicks, and Fink, 1978). This first success-ful transformation of a eukaryotic cell marked a break-through in (yeast) molecular biology. Several types ofepisomal (designated YEp), highly replicating (YRp), or chro-mosomally integrating shuttle vectors (YIp) were designed,carrying various selectable markers and/or various elementsallowing the expression of particular yeast – or even foreign –

genes (Broach, Strathern, and Hicks, 1979; Struhl et al.,1979; Hadfield, 1994). They thus became widely applicablefor studying single genetic entities or for reverse geneticapproaches. Of particular interest was the notion that a chi-meric plasmid endowed with a segment of centromericDNA (YCp) and transformed in yeast cells stabilizes thisplasmid. During mitotic cell division, this plasmid will benormally replicated once, and the two copies are segregatedbetween mother and daughter cells in a 1 : 1 ratio. In meioticdivision, the four copies will be segregated at a 2 : 2 ratio.Hundreds of different vectors of these types have beendevised to date and have been made commercially available.

4.2.2.1 Yeast Shuttle VectorsGenerally, the plasmid vectors (“shuttle vectors”) containgenetic material derived from the Escherichia coli vectorpBR322 (or its derivatives), and a genetic element (origin ofreplication) that enables them to be propagated in E. coli cellsprior to transformation into yeast cells and a selectablemarker (mainly the b-lactamase gene, amp) for the bacterialhost (Figure 4.1).

Additionally, the shuttle vectors harbor a selectable marker(Table 4.2 and Figure 4.2) to be used in the yeast system.Conventionally, markers are genes encoding enzymes forthe synthesis of a particular amino acid or nucleotide, so thatcells carrying the corresponding genomic deletion (or muta-tion) are complemented for auxotrophy or autotrophy.Further, these vectors contain a sequence of (combined)restriction sites (multiple cloning site, MCS) that will allowcloning of foreign DNA into this locus. Convenient markersdeveloped for the screening of large collections of mutantcells are the lacZ gene or the kanamycin-resistance gene(kan) gene (Wach et al., 1994a). The chloramphenicol-resistance gene (cat) (Mannhaupt et al., 1988) or the fireflyluciferase gene (Gould and Subramani, 1988; Contag and

Fig. 4.1 Yeast shuttle vectors.

Table 4.2 Markers used in yeast recombinant DNA technology.

Markertype

Gene Comment

Recessive LEU2, TRP1LYS2, HIS3

genes complementing auxotrophicmutations for amino acidbiosynthesis

URA3, ADE2 genes complementing autotrophyfor nucleotides

lacZ b-galactosidase from E. coliamyloglucosidase enzyme activity used for screening

rather than for selection

luciferaseDominant CUP1 copper resistance

G418 aminoglycoside resistanceTUN tunicamycin resistanceKAN kanamycin resistancehygromycin genes conferring resistance to

drugschloramphenicolcanavanine

4.2 Genetic Engineering and Reverse Geneticsj61

Bachmann, 2002; Massoud et al., 2007) can be integratedinto vectors in combination with promoter sequences fromyeast to monitor expression levels. Promega rendered a pro-tocol on bioluminescence assays in 2009 (http://www.promega.com/multimedia/bioLum01.htm).

Principally, four types of shuttle vectors can be distin-guished (Figure 4.1) by the absence or presence of additionalgenetic elements:

� Integrative plasmids (YIp), which by homologousrecombination are integrated into the host genome at thelocus of the marker, when this is opened by restriction andlinearized DNA is used for transformation. This (nor-mally) results in the presence of one copy of the foreignDNA inserted at this particular site.

� Episomal plasmids (YEp), which carry part of the 2mmplasmid DNA sequence necessary for autonomous replica-tion. Multiple copies of the transformed plasmid are prop-agated in the yeast cell and maintained as episomes.

� Autonomously replicating plasmids (YRp), which carry ayeast origin of replication (autonomous replicatingsequence (ARS) sequence) that allows the transformedplasmids to be propagated several 100-fold.

� Centromeric plasmids (YCp). In addition to an ARSsequence these vectors carry a centromeric sequence(derived from one of the nuclear chromosomes) that nor-mally guarantees stable mitotic segregation and reducesthe copy number of self-replicated plasmid to just one.

Numerous biochemical companies offer collections ofcanonical and newly developed yeast vectors with appropriatemarkers, together with relevant information or referencesfor application.

Three main methods are used in yeast cell transformation.(i) Permeabilization of the cells by the use of lithium acetate(Ito et al., 1983). (ii) Electroporation of yeast cells using spe-cial devices (Neumann et al., 1982; Weaver and Chizmadz-hev, 1996). Explanations and pictures of equipment canbe found at http://en.wikipedia.org/wiki/Electroporation.(iii) Bombardment of cells with DNA-coated tungsten orgold microprojectile particles (Taylor and Fauquet, 2002).Desired genetic material is precipitated onto micron-sizedmetal particles and placed within one of a variety ofdevices (“gene guns”) designed to accelerate these micro-carriers to velocities required to penetrate the cell wall. Inthis manner, transgenes can be delivered into the cell’sgenome. Since the late 1980s microparticle bombardmenthas become a powerful tool for the study of gene expres-sion and production of stably transformed tissues andwhole transgenic organisms for experimental purposesand practical applications.

4.2.2.2 Yeast Expression VectorsYeast expression vectors employ promoter and terminatorsequences in addition to the gene of interest (inserted in thecorrect reading frame). It is advantageous to use yeast-derived (homologous) rather than heterologous regulatorysequences, because the former are more efficient and heter-ologous elements will sometimes not work in yeast. Table 4.3lists some of the promoter modules that are in use. Constitu-tive promoters are derived from genes of the glycolytic path-way, because these lead to high-level transcriptionalexpression.

On the other hand, regulated promoters can be controlledby controlling the availability of certain nutrients. This allowsaugmenting yeast cell mass prior to heterologous geneexpression, so that the cell population can be optimizedbefore the regulated promoters are turned on.

Fig. 4.2 Processing of proteins fused to MFa1 of yeast.

Table 4.3 Regulated promoter elements in yeast expression vectors.

Promoter type Gene module Encoded protein Strength Regulation

Constitutive ADH1 alcohol dehydrogenase 1 þþþPGK1 phosphoglycerate kinase þþþþ 10-fold induction by glucoseENO enolasePYK1 pyruvate kinase þþþ 20-fold induction by glucose

Regulated GAL1 galactose enzymes þþþ 1000-fold induction by galactoseGAL7GAL10ADH2 alcohol dehydrogenase 2 þþ 100-fold repressed by glucosePHO5 acid phosphatase þþ 200-fold repressed by phosphateMET25 O-acetyl homoserine sulfhydrylase þCUP1 copper metallothionein þ 20-fold induced by Cu2þ

Heterologous CaMV cauliflower mosaic virus 35S promoterGRE glucocorticoid response elementARE androgen response element

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4.2.2.3 Secretion of Heterologous Proteins from YeastProtein secretion in yeast is a complex process and there isno generally accepted signal sequence that directs secretion.Although several foreign proteins can be secreted under thedirection of their own signals, homologous signal sequencesare much more successful and can result in highly expressedheterologous proteins recoverable from the extracellularmedium.

Frequently used signal sequences in Saccharomyces cerevi-siae include those derived from invertase (SUC2), acid phos-phatase (PHO5), or a-factor pheromone (MFa1; Figure 4.2).It is of value that the specificity of the signal processingenzymes for the a-factor precursors allows for the produc-tion of heterologous proteins with authentic N-termini(Brake, 1989).

The prepeptide directs secretion of the protein into the ERand is removed by the signal peptidase. The propeptide iscleaved in the Golgi apparatus by the Kex2 endopeptidase,which cuts C-terminally to Lys–Arg. This leaves the two Glu–Ala peptides attached to the N-terminus of the mature pro-tein. These are serially removed by Ste13 exopeptidase diges-tion. The possible sites for heterologous protein fusion to theMFa1 leader are indicated. The three glycosylation sites aremarked by encircled Gs.

Other important molecular aspects of recombinant pro-teins expressed in yeast are the features of post-translationalprocessing and modification processes specific to yeast, par-ticularly with attention to therapeutic agents produced inyeast. N- and O-linked glycosylation patterns in yeast mayprove to be different from those in the native host. For exam-ple, yeast adds mannose units to threonine or serine resi-dues, while higher eukaryotes prefer sialic acid O-linked sidechains. Such differences may affect the folding, stability,activity, and immunogenicity of proteins produced in yeast.By contrast, N-linked glycosylation in yeast largely resemblesthat of higher eukaryotes. Attention has also to be paid topossible differences in phosphorylation, acetylation, methyl-ation, myristoylation, and isoprenylation of proteins in yeastin relation to other organisms.

Once synthesized and modified, heterologous proteinsproduced in yeast may undergo intracellular proteolyticdegradation before they can be purified. In S. cerevisiae,proteolysis may be unspecific and associated with the vac-uole, or specific and coupled to the ubiquitin–proteasomesystem.

4.2.2.4 Fluorescent Proteins Fused to Yeast ProteinsA relatively recent development of labeling proteins involvesthe Green Fluorescent Protein (GFP) from the jellyfish(Aequorea victoria) as a reporter molecule (Prasher et al.,1992), as well as several derivatives of GFP with fluorescencespectra shifted to other wavelengths (Heim et al., 1994;Heim, Cubitt, and Tsien, 1995). Fusions of genes of interestwith the fluorescent protein gene (N- or C-terminal) alsoallow to follow the expression and destiny of the fusion pro-teins followed by fluorescence microscopy (Niedenthal et al.,

1996; Wach et al., 1997; Hoepfner et al., 2000). Fusion pro-teins with the conventional GFP moiety (some 200 aminoacids in length) can be visualized by fluorescence micros-copy at 395 nm (blue light). Interestingly, four variants ofGFP, having particular amino acid replacements, becameavailable that emit fluorescent light of lower (red) or higher(blue) wavelengths. These “mutant” GFPs are: EnhancedGFP (EGFP; S65T) with brighter performance (Heim,Cubitt, and Tsien, 1995), Enhanced Blue Fluorescent Protein(Y66H), Enhanced Cyan Fluorescent Protein (Y66W), andYellow Fluorescent Protein (T203Y). A nice review on thedevelopment of the fluorescent dyes is presented in theNobel Lecture of Roger Tsien (Tsien, 2008).

In most cases, the globular extension in the modifiedprotein will not influence its intracellular localization orits function as compared to the native protein, indepen-dent of whether the GFP moiety has been fused to the N-or C-terminus. However, this has to be checked individu-ally for each protein of interest. Variants of the EGFPbecame commercially available, the genes of which havebeen modified such that they are adapted to codon usagein plants, and these have also proven to be advantageousin expression in the yeast system. Figure 4.3 shows twosuch vectors that were used for multiple expression stud-ies of various yeast proteins (Mannhaupt and Feldmann,unpublished).

Fig. 4.3 Vectors with GFP cassettes for fluorescent yeast fusion proteins.

4.2 Genetic Engineering and Reverse Geneticsj63

In a breakthrough discovery, Matz et al. (1999), isolated agene encoding a Red Fluorescent Protein (“DsRed”) from acoral (Discosoma sp.) in a Moscow aquarium. Later, Campbellet al. (2002) succeeded in generating a monomeric Red Fluo-rescent Protein, since the native molecule had been isolatedas a tetramer. This Red Fluorescent Protein was furtherimproved by engineering a broad series of at least 11 differ-ent (mutant) Red Fluorescent Proteins with different absorp-tion and emission maxima covering the rest of the visiblespectrum (Shaner et al., 2004).

4.2.3Yeast Cosmid Vectors

Cosmid vectors have proven to be very convenient forcloning and sequencing of large segments of yeast chro-mosomal DNA. To construct a library with as completecoverage as possible with as few clones as possible, thecloned DNA fragments should be randomly distributed onthe DNA. Under these conditions, the number of clones(N) in a library representing each genomic segment with agiven probability (P) is:

N ¼ lnð1� PÞ=lnð1� f Þ

where f is the insert length expressed as fraction of thegenome size (Clarke and Carbon, 1976) (Table 4.4). Forexample, with a size of 12 800 kb for the yeast genome andassuming an average insert length of 35 kb, a cosmidlibrary containing 4600 random clones would represent theyeast genome at P¼ 99.99% (i.e., about 12 times thegenome equivalent). The actual number of cosmid clonesobtained by the usual procedures is very high (more than200 000/mg DNA).

One of the first yeast cosmid vectors, pHC79, was devel-oped in 1980 (Hohn and Collins, 1980). In connection withthe Yeast Genome Sequencing Program, two major types ofcosmids have been employed (Figure 4.4):

i) pYc3030 generated from pCH79 by adding the yeast2mm plasmid origin of replication and the yeast HIS3marker is a shuttle vector that most convenientlyallows DNA to be shuttled between E. coli and yeastcells (Stucka and Feldmann, 1994). It contains aBamHI cloning site, which is suitable for accommo-dating yeast DNA fragments of about 30–45 kb in sizeobtained by partial digestion of high-molecular-weightDNA with Sau3A. For cloning, the vector arms com-prising the l phage cos sites have to be preparedseparately and are ligated to a mixture of partialSau3A fragments that have been size-fractionated bycentrifugation of the digestion mixture in NaCl gra-dients. Replica plating – one of the common proce-dures used for the storage and screening of cosmidlibraries – has been successfully applied to yeast cos-mid libraries. Colonies can be easily purified, andcosmid DNA can be prepared by one of the “mini-prep” procedures. We found that yeast cosmid canbe stored at �20 �C for several years without dam-age. Cosmids have not only been used successfullyfor chromosomal walking, but also in complementa-tion analyses; cosmids are maintained in yeast cellsin only one or a few copies.

ii) pWE15 (and pWE16) are cosmid vectors that havebeen designed for genomic walking and rapid restric-tion mapping (Thierry et al., 1995). They contain bacte-riophage T3 and T7 promoters, respectively, flanking aunique BamHI cloning site. By using the cosmid DNAcontaining a genomic insert as a template for either T3or T7 polymerase, directional “walking” probes can besynthesized and used to screen genomic cosmid libra-ries (or sublibraries). These vectors contain additionalgenes (SV2-neo or SV2-dhfr, respectively), which allowthe expression, amplification, and rescue of cosmidsin mammalian cells. NotI restriction sites have beenplaced near the BamHI site, which allows the insert tobe removed as a single large fragment.

Table 4.4 Number of cosmids covering yeast chromosomes at differentprobabilities.

Chromosome Size(kb)

Number ofcosmids P¼ 99%

Number of cosmidsP¼ 99.999%

I 220 26 80II 840 108 324III 320 39 119IV 1600 208 624V 610 78 233VI 280 35 103VII 1200 155 601VIII 560 71 214IX 450 57 170X 760 98 293XI 670 79 258XII 2200 288 861XIII 920 119 356XIV 800 103 309XV 1110 142 427XVI 960 124 372

Fig. 4.4 Examples of yeast cosmid vectors.

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4.2.4Yeast Artificial Chromosomes

The construction of yeast artificial chromosomes (YACs) fol-lows a similar strategy to that of the ARS/CEN (centromere)plasmids (Burke, Carle, and Olson, 1987). In addition to theusual components, they are endowed with telomeresequences flanking a yeast marker gene (HIS3 in pYAC4;Figure 4.5); restriction sites flanking the telomere sequencescan later be used to linearize the plasmid DNA for yeasttransformation. The insertion site for large foreign DNA seg-ments is located within a second “marker” gene, the SUP4gene encoding a suppressor tRNA, which allows selection oftransformed cells that possess the appropriate genetic back-ground. As the linearized plasmids behave like endogenouschromosomes, they are maintained and replicated in thesame manner as resident yeast chromosomes. A prerequisitein this approach was the electrophoretic karyotyping of yeast(Carle and Olson, 1985). In the beginning, when applied tohuman DNA, the YACs were of considerable advantage formapping human chromosomes. Unfortunately, the high pro-pensity of yeast to recombination via short (around 70-bp)homology regions resulted in too many mapping failures.This caveat in the use of YACs, which has been noticed par-ticularly in conjunction with the Human Genome Project,has led to the development of other methods for mappinglarge genomes.

4.3More Genetic Tools from Yeast Cells

4.3.1Yeast Two-Hybrid System

A novel technique revolutionizing the detection of protein–protein interactions of any kind was established by Fieldsand Song (1989). The yeast two-hybrid system has beendeveloped as a potent tool to identify cDNAs, carried on one

plasmid, which code for proteins that interact with a targetprotein specified by a DNA sequence carried on anotherplasmid. This simple approach was based on the uniqueproperties of the yeast Gal4p transcriptional activator regulat-ing the expression of GAL4 and hence other galactose genesin yeast (see Chapter 10); the Gal4p transcriptional activatoris composed of two physically separable, functionally inde-pendent activation and binding domains (Gal4-AD and Gal4-BD, respectively). The cloning vectors, which are endowedwith different markers, are used to create fusions of theGAL4 domains with genes for proteins that potentially inter-act. After introduction of these entities into a yeast strain thatcarries an appropriate reporter gene (HIS3 or lacZ) with aGAL4 upstream activating sequence (UAS) element in itspromoter, only upon interaction of the two domains theDNA-BD will be tethered to the AD and will reconstitute theGal4p transcriptional activator, which then results in the acti-vation of the reporter gene (Figure 4.6). A selection of posi-tive clones can be achieved by screening them for Hisþ aswell as LacZþ positives, and the GAL4-AD/library fusionplasmid can efficiently be retrieved from such colonies. Themethod has been improved since its invention (Martzenet al., 1999), particularly to minimize the appearance of falsepositives, which, however, still seems to be a problem notcompletely overcome. In addition, a yeast three-hybrid sys-tem for detecting small ligand–protein receptor interactionswas developed in the late 1990s (Licitra and Liu, 1996; Hooket al., 2005). Bacterial two- and n-hybrid systems later cameinto use as well (Hu, Kornacker, and Hochschild, 2000; Doveand Hochschild, 2004).

In the past decades, the two-hybrid system has beenwidely used to detect protein–protein interactions in yeast aswell as for proteins from other organisms, even in theirnative environment. Meanwhile this approach, which peoplewere initially reticent to apply, has now been approved forlarge-scale and high-throughput protocols. However, withthe massive application of this method in systems biology,some limitations have become apparent. Readers are invitedto solicit the help of an article by Br€uckner et al. (2009) thatprovides an overview on available yeast two-hybrid methods,in particular focusing on more recent approaches. Detection

Fig. 4.5 YACs.

Fig. 4.6 Principle of the yeast two-hybrid system.

4.3 More Genetic Tools from Yeast Cellsj65

of protein interactions in their native location (e.g., in thecytosol or bound to a membrane) is made possible by theuse of cytosolic signaling cascades or split protein constructs.Strengths and weaknesses of these genetic methods are dis-cussed and some guidelines for verification of detected pro-tein–protein interactions are emphasized (Br€uckner et al.,2009). Two-hybrid approaches are very labor intensive. Someof the most comprehensive protein–protein binary interac-tion data in S. cerevisiae were obtained by Yu et al. (2008a), byperforming high-throughput yeast two-hybrid screeningwith 3917 bait proteins and 5246 prey proteins, whichyielded 1809 interactions among 1278 proteins. Ninety-fourrandomly chosen interactions were validated with a preci-sion rate of 94–100% in that study. The results of some otherlarge-scale two-hybrid studies in yeast can be found inSection 12.3.4.

Since its initial development, the two-hybrid system hasbeen adapted to the use of further baits and methods to mon-itor the outcome (Table 4.5).

4.3.2Yeast Three-Hybrid System

An elegant extension of the yeast two-hybrid system wasbuilt to examine RNA–protein interactions (SenGupta,Wickens, and Fields, 1999; Wurster and Mahler, 2010); thatis, to identify RNA ligands for an RNA-binding protein. Aprotein–RNA interaction is detected by the reconstitution ofa transcriptional activator (Gal4D) using two hybrid proteinsand a hybrid RNA. The tethering of the RNA molecule isachieved to the promoter of a reporter gene (which, forexample, uses the LexA promoter together with lacZ orHIS3as in the two-hybrid approach) by binding it to a hybrid pro-tein consisting of the bacteriophage MS2 coat protein fusedto the DNA-binding protein LexA. The RNA-binding domain

(red hairpin in Figure 4.7) to be analyzed is fused to an RNAmolecule that binds to the MS2 coat protein (black hairpin),thus rendering a “bifunctional” RNA (“bait”) that mediatesthe contact between the Lex promoter on the one end and toa “prey” protein (green) associated with the transcriptionalactivation domain of yeast Gal4p (yellow) at the other end.

In the first test system, the authors checked an RNAlibrary that was built by transcribing short genomic yeastDNA fragments together with binding sites for the coat pro-tein. This hybrid RNA library was then screened for RNAsthat bound to the yeast Snp1p protein and yielded as thestrongest positive the fragment of U1 RNA that containsloop I, which is known to bind to Snp1 in U1 small nuclearribonucleoprotein (snRNP). Similarly, four other RNAligands were detected that produced weaker three-hybridsignals, suggesting lower affinities for Snp1 compared to U1RNA. In addition, this search also yielded a set of RNAsequences that can activate transcription on their own whenbound to a promoter through a protein interaction.

Table 4.5 Overview of different yeast two-hybrid approaches.

Method Possible baits Response Executingcompartment

Reference

Classic yeast two-hybrid nontransactivating proteinscapable of entering nucleus

transcriptional activation nucleus Fields and Song, 1989

SOS recruitment transactivating cytosolic proteins RAS signaling membrane Aronheim et al., 1994Membrane split ubiquitinsystem

membrane proteins transcriptional activation membrane Stagljar et al., 1998

Ras recruitment system transactivating cytosolic proteins RAS signaling membrane Broder, Katz, andAronheim, 1998

Dual-bait system two nontransactivating proteinscapable of entering nucleus

transcriptional activation nucleus Serebriiskii et al., 1999

G-protein fusion membrane proteins inhibition of G-proteinsignaling

membrane Ehrhard et al., 2000

RNA polymerase III-based yeast two-hybrid

transactivating proteins inpolymerase III pathway

inhibition of transcriptionalactivation

nucleus Petrascheck, Castagna,and Barberis, 2001

SCINEX-P system extracellular and transmembraneproteins

downstream signaling andtranscriptional activation

ER Urech, Lichtlen, andBarberis, 2003

Cytosolic split ubiquitinsystem

transactivating, cytosolic proteins transcriptional activation ERmembrane

Mockli et al., 2007

Fig. 4.7 Principle of the yeast three-hybrid system.

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4.3.3Yeast One-Hybrid (Matchmaker) System

The yeast one-hybrid system provides the basic tool for con-ducting a one-hybrid assay – an in vitro genetic assay used forisolating novel genes encoding proteins that bind to a target,cis-regulatory element, or any other short, DNA-bindingsequence. The one-hybrid assay offers maximal sensitivitybecause detection of the DNA–protein interactions occurswhile proteins are in their native configurations in vivo. Inaddition, the gene encoding the DNA-binding protein ofinterest is immediately available after a library screening.Figure 4.7 presents a specific example, in which the tran-scription factor Rpn4p interacting with a particular UAS ele-ment (PACE) has been cloned (Mannhaupt et al., 1999).

To conduct a one-hybrid assay, it seems useful first to gen-erate the reporter yeast strains. Tandem copies of a poten-tially regulatory DNA element are inserted upstream of aHIS3 and/or lacZ reporter gene promoter in an integratingvector. Cells transformed with these constructs and testedfor their site-specific integration can than be propagated andused as reporter strains. Expression of HIS3 or lacZ in thesestrains is under the control of the potential DNA-bindingmotif. The reporter strain is then transformed with plas-mids that contain random yeast DNA fragments precededby the GAL4 binding domain under the control of a strongpromoter. For example, a GAL4-AD library can bescreened for a cDNA encoding the DNA-binding proteinof interest. Positive transformants can be selected eitherby growing transformants on minimal medium lackinghistidine or on X-gal plates in Figure 4.8a. If a HIS3/lacZreporter strain has been used, a b-galactosidase assay canbe performed to verify the DNA–protein interaction andhelp eliminate false positives. The constructs to be testedshould contain a copy of the gene identified to encode theDNA-binding protein expressed under a strong promoter(e.g., TDH3; Figure 4.8b). It is possible to fuse the GFPmoiety to the gene in question. The b-galactosidase assaycan be conducted as an “overlay” test.

4.4Techniques in Yeast Genome Analyses

4.4.1Microarrays

In the late 1990s, microarrays were invented as a convenientmethodology for the investigation of large numbers of sam-ples (e.g., on a genomic scale) – so-called “high throughputanalyses” (DeRisi, Iyer, and Brown, 1997). The purifiedprobes were arrayed with a 48-pin electrospray ionization(ESI) contact printer on an appropriate support surface:nanowells or solid surfaces, such as chemically modified orcoated glass microscope (1.8 cm � 1.8 cm) slides, or nitro-cellulose- or amino-silane-coated slides. Readout of the latter

formats (Figure 4.9) could be achieved with many types ofcommercial scanners.

4.4.1.1 DNA-Based ApproachesInitially, hundreds to thousands of DNA samples wereaccommodated on microarrays, which then could be probedwith different tags. Preparation of the spotted samples wasperformed by two principal approaches, depending on thepurpose of the respective study.

Fig. 4.8 Example of the yeast one-hybrid system (see text for explanation).

Fig. 4.9 Schematic view of a microarray.

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(i) For the construction of genome-wide deletion mutants,a common procedure was applied, which was based on theautomated deletion of each single open reading frame (ORF)and a single-step gene replacement strategy, introducing thekanamycin-resistance gene as a marker instead of the geneof interest via homologous recombination at the flankingregions of this gene and replacing it by a unique 20-bpsequence to serve as a “barcode” (Winzeler et al., 1999); themethod is outlined in Figure 4.10. (ii) For hybridizationexperiments, each single gene was amplified automaticallyby the polymerase chain reaction (PCR) technique and thecomplete collection of these ORFs fixed on microarrays.Thus, it became feasible to accommodate the entire set ofyeast genes (6400 in an 3.24 cm2 array of 80� 80 spots) toone chip, which could be simultaneously hybridized withtwo full complements of differently fluorescently labeledmRNAs – one derived under “standard” conditions and usedas a reference, while the other one is used to monitorchanges in expression profiles under varying biologicalparameters, such as growth conditions (cell states, media),stress conditions, particular deletants, or overexpressants(“master” genes, transcription factors, etc.). Differentlabeling of mRNAs or cDNAs derived from these wereachieved by fluorescent dyes with emissions of differentwavelengths. Several routines were developed for the eval-uation and documentation of the results obtained bymicroarray techniques. The majority of these studies aredocumented in Section 12.3.

As an example, a partial profile of gene expression duringyeast sporulation (Chu et al., 1998) is modeled in Figure 4.11.Genes correspond to the lines and the time points of eachexperiment are the columns. The ratio of induction/repres-sion is shown for each gene such that the magnitude is indi-cated by the intensity of the colors displayed. If the color isblack then the ratio of control to experimental cDNA is equalto 1, while the brightest colors (red and green) represent aratio of 2.8 : 1. Ratios greater than 2.8 are displayed as thebrightest color. In all cases red indicates an increase inmRNA abundance while green indicates a decrease in

abundance. Gray areas (when visible) indicate absent data(or data of low quality). Blue bars on the side of the figureindicate matching of the consensus sequence of an upstreamregulatory element (upstream repression sequence 1 (URS1)or mid-sporulation element (MSE); cf. Section 7.3.1); thebrighter the color, the more stringent the match.

4.4.1.2 Proteome AnalysesUntil relatively recently, investigation of the full proteome hadbeen an intimidating task. In addition to the incomplete defi-nition of the proteome, the technical limitations for large-scaleprofiling of proteins were enormous. However, during thepast years, improved and novel technologies have emerged aspowerful tools for proteomic studies, including shotgun prote-omics by mass spectrometry (MS) technology (Wu and Han,2006) and protein microarray technology (Zhu et al., 2001). Incombination, these two approaches have been used exten-sively in biological research such as proteome profiling, pro-tein–protein interaction mapping, and identification of post-translational modifications (Chen and Snyder, 2010). Thegreatest advantage of this technology lies in its capability ofhigh-throughput protein identification and quantification.

Normally, a protein microarray contains hundreds to thou-sands of proteins arrayed in an interpretable format(cf. Figure 4.9). Two types can be distinguished: analytical(or diagnostic) microarrays and functional microarrays.One form of diagnostic microarray is the antibody micro-array, in which specific antibodies against defined target pro-teins are arrayed on the surface of a support material (e.g.,glass slides); they are used for the detection and quantifica-tion of specific antigens.

A functional microarray is usually set up from a largenumber of individually expressed and purified functional,

Fig. 4.10 Tagging yeast genes by a “barcode” sequence in a one-step gene

replacement strategy.

Fig. 4.11 Model for the interpretation of gene expression (see text for

explanation).

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full-length proteins or peptides printed in a high-density for-mat on support surfaces, and can represent the complete orpartial proteome of a given organism; for S. cerevisiae, about6400 spots are sufficient to represent the whole proteome.This type of protein microarray has been employed in stud-ies of protein–protein, protein–DNA, and protein–smallmolecule interactions as well as protein modifications(Figure 4.12). Manufacturing a protein microarray startswith preparing collections of full-length yeast genes clonedin expression plasmids that produce either N-terminaltagged (e.g., glutathione-S-transferase (GST)) or C-terminaltagged (e.g., tandem affinity purification (TAP)) fusion pro-teins. The recombinant yeast proteins are expressed in indi-vidual yeast clones in a 96-well format and purified usingthe corresponding affinity tags (e.g., GST or TAP). The purif-ied proteins are then arrayed with a 48-pin ESI contactprinter on appropriate support surface: these may be nano-wells or solid surfaces (such as chemically modified orcoated glass microscope slides, or nitrocellulose- or amino-silane-coated slides. These latter formats will be compatiblewith many commercial scanners. Detailed manufacturers’protocols can be found in Fasolo and Snyder (2009). For pro-tein–protein, protein–DNA and protein–small-moleculeinteraction studies, these arrays are usually probed with fluo-rescently labeled molecules and the signals are thenacquired with a confocal laser scanner (cf. Figure 4.9).

Compared to the two-hybrid approach for identifying pro-tein–protein interactions, the protein microarray has manyadvantages for this purpose. (i) Oncemanufactured, its in vitronature does not require yeast culture, transformation, andmating, which greatly saves time and effort. (ii) Fluorescently

labeled probes are used instead of reporter genes, so that therelative fluorescence intensity can reflect the binding strengthof the two interacting proteins and the interaction signal canbe readily quantified with a laser scanner.

Post-translational modifications (including phosphoryl-ation, glycosylation, acetylation, ubiquitination, SUMOylation,and S-nitrosylation), which ultimately form functional accesso-ries, are also amenable to detection by microarray techniques.In some respects, the status of post-translational modificationof the proteome is a snapshot of the dynamic activities of theliving cell. For example, a comprehensive screening of 119 outof the 122 yeast kinases with 17 different substrates yielded amagnificent overview on the cellular activities of the kinases(Mok, Im, and Snyder, 2009). The substrateswere immobilizedonto nanowell protein chips and phosphorylation events wereidentified by adding [g-33P]ATP and a specific yeast kinase, andexposing the chip to a phosphoimager.

Expectedly, protein microarray technology also has limita-tions. In the first place, its meaningfulness relies heavily onthe accessibility of genomic information of the respectiveorganism. Microarrays are not capable of covering unknownORFs or splicing variants and are unavailable for organismswhose genome information is unknown. In higher orga-nisms, a limitation is that for most genes usually only onesplicing variant is used to represent the specific gene, there-fore the splicing diversity of the proteome is under-repre-sented. This problem is limited in yeast, because only 5% ofthe genes possess introns. A second drawback is that pro-teins are purified from the cells, so they may contain mixedpost-translational modifications or even copurified interact-ing proteins, which disturb the picture.

Fig. 4.12 Applications of functional protein

microarrays. (Modified from Fasolo and

Snyder, 2009.)

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4.4.2Affinity Purification

Isolation and identification of protein partners in multipro-tein complexes are important in gaining further insightsinto the cellular roles of proteins and determining the possi-ble mechanisms by which proteins have an effect in themolecular environment. A former useful method for theidentification of new protein–protein interactions consistedof a screening procedure by pull-down experiments withGST fusion proteins attached to glutathione beads. Thesepull-downs were often considered a necessary complementa-tion for two-hybrid results and could easily be coupled withMS (Brymora, Valova, and Robinson, 2004).

The TAP method was originally developed in yeast for thepurification of protein complexes and the identification ofprotein–protein interactions (Puig et al., 2001). The methodaimed at rapid purification of proteins to near homogeneityand under native conditions. The TAP tag (N- or C-terminallyfused) contains two adjacent affinity purification tags (acalmodulin-binding peptide and Staphylococcus aureusProtein A) separated by a tobacco etch virus (TEV) proteasecleavage site. During the first purification step, the Protein Amoiety of the TAP tag is bound to IgG beads and proteincomponents associated with the TAP-tagged protein areretrieved by TEV protease cleavage. In the second affinitystep, the protein complex is immobilized to calmodulin-coated beads via the calmodulin-binding peptide of the TAPtag. Variations of the method to specifically purify complexescontaining two given components or to subtract undesiredcomplexes can easily be implemented. Recent developmentsin sample preparation and affinity purification strategiesallow the capture, identification, and quantification of pro-tein interactions of protein complexes that are stable,dynamic, transient, and/or weak.

4.4.3Mass Spectrometry

Affinity purification coupled with quantitative MS hasbecome the primary method for studying in vivo proteininteractions of protein complexes and whole-organism pro-teomes. There are various protocols based on stable isotopelabeling for protein quantitation, such as SILAC (stable iso-tope labeling by amino acids in cell culture) (Ong et al.,2002; Emadali et al., 2009), ICAT (isotope-coded affinity tags)(Gygi et al., 1999), ICPL (isotope-coded protein labels)(Schmidt, Kellermann, and Lottspeich, 2005; Lottspeich andKellermann, 2011), and iTRAQ (amine-reactive isobaric tag-ging reagents) (Ross et al., 2004).

SILAC was originally applied for in vivo incorporation ofspecific amino acids into all mammalian proteins. Mamma-lian cell lines can be grown in media lacking a standardessential amino acid (e.g., leucine), but supplemented with anonradioactive, isotopically labeled form of it, in this casedeuterated leucine (Leu-d3). This treatment does not changethe growth from that in normal media. Complete

incorporation of Leu-d3 occurs after five doublings in thecell lines and proteins studied. Protein populations fromexperimental and control samples can be mixed directly afterharvesting and MS identification is straightforward as everyleucine-containing peptide incorporates either all normalleucine or all Leu-d3.

For ICAT (Gygi et al., 1999), chemical probes are used thatconsist of three general elements: a reactive group labeling adefined amino acid side chain (e.g., iodoacetamide to modifycysteine residues), an isotopically coded linker, and a tag(e.g., biotin) to allow the affinity isolation of labeledproteins/peptides. For the quantitative comparison of twoproteomes, one sample is labeled with the isotopically light(d0) probe and the other with the isotopically heavy (d8)version. Both preparations are then combined, digested witha protease (i.e., trypsin), and subjected to avidin affinity chro-matography to isolate peptides labeled with isotope-codedtagging reagents. These peptides are analyzed by liquidchromatography/MS. The ratios of signal intensities ofdifferentially mass-tagged peptide pairs are quantified todetermine the relative levels of proteins in the two prepara-tions. The original tags were developed using deuterium,but later it was possible to use 13C instead to circumventissues of peak separation during MS due to the deuteriuminteracting with the stationary phase of the column.

ICLP (Lottspeich and Kellermann, 2011) is aimed at thequantitative analysis of even low abundant proteins. There-fore, it is indispensable to reduce complexity on the level ofproteins by several fractionation steps. To compensate forthese time-consuming steps and to avoid nonreproducibleloss of protein species, isotope labeling with “ICPL Quadru-plex” is the method of choice to achieve confident results.The method is based on stable isotope tagging at the freeamino groups of intact proteins. After labeling of up to fourdifferent proteome states the samples can be combined andthe complexity reduced by any separation method presentlyemployed in protein chemistry. After enzymatic cleavage ofthe protein fractions, the ratios of peptides in the differentproteome states can be calculated by simple MS-based massspectrometric analyses. Only peptides that exhibit regula-tions in the different proteome states are further investigatedfor identification by tandem MS (MS/MS). The quantifica-tion of multiplexed ICPL experiments is greatly facilitated bythe recently published ICPLQuant software, which includesa complete peptide database for comparisons.

The experimental sampling is done as follows. Four pro-tein mixtures obtained from four distinct cell states, tissues,or body fluids are individually reduced and alkylated todenature the proteins, and to ensure easier access to freeamino groups. These samples are labeled each with one ofthe four ICPL reagents (ICPL0, ICPL4, ICPL6, and ICPL10).After combining the mixtures, complexity is reduced by theaforementioned measures. Quantification and identificationis done by high-throughput MS. Since peptides with an iden-tical amino acid sequence derived from the four differentiallylabeled protein samples differ in mass, they appear as quad-ruplets in the acquired MS spectra. The relative abundance

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of their parent proteins in the original samples can be deter-mined from the ratios of the ion intensities of these sisterpeptide pairs. It is recommended to run reference samples,which are prepared similar to the original samples.

iTRAQ, the multiplexed protein quantitation strategy, pro-vides relative and absolute measurements of proteins incomplex mixtures. This methodology is centered to a multi-plexed set of isobaric reagents that yield amine-derivatizedpeptides. The derivatized peptides are indistinguishable inMS, but exhibit intense low-mass MS/MS signature ionsthat support quantitation. In their study, Ross et al. examinedthe global protein expression of a wild-type yeast strain and

the two isogenic Dupf1 and Dxrn1 mutant strains that aredefective in the nonsense-mediated mRNA decay and thegeneral 50 ! 30 decay pathways, respectively.

Recently, technological developments in MS-based proteo-mics approaches have made comprehensive characterizationof protein complexes possible by enabling the determinationof dynamic protein complex compositions, stoichiometries,post-translational modifications, assemblies, structures, andprotein interaction networks. With the development of newaffinity tags and antibodies, affinity purification/MS-basedstrategies have taken center stage for the study of multisubu-nit protein complexes.

Summary

� Initially, some conventional methodology is presented(i.e., how yeast cells can be grown and opened in order toisolate particular subcellular constituents). In the presentlaboratory routine, these methods might no longer beapplied. A description of genetic techniques (e.g., geneticmapping, genetic crosses, or tetrad analysis) has beenexcluded a priori. Preparation of recombinant DNA andtransformation of yeast cells are still practiced in many lab-oratories. As this volume does not aim at describing theprotocols for the various procedures, we just explain theprinciples and refer the reader to the relevant literature.Useful information on equipment, growth media, appro-priate yeast vectors, and strains can frequently be found inthe brochures or catalogs of the relevant companies.

� The successful transformation of yeast cells by hybridplasmids in 1978 marked a milestone in molecular biology.Depending on their molecular shaping, these plasmidswould autonomously replicate in yeast (as single or multi-copy entities) or integrate as single copies into defined lociwithin the yeast genome. A plasmid endowed with a seg-ment of centromeric DNA and transformed in yeast cells isstabilized; during mitotic cell division this plasmid will benormally replicated once and the two copies segregatedaccording to the rules known for the yeast chromosomes.Shuttle vectors, capable of propagating both in yeast and inbacterial cells, allowed reciprocal transfer of genetic mate-rial from one host to the other. Selection of transformedcells was facilitated by inclusion of appropriate geneticmarkers into the plasmid sequences. Replica plating ofyeast cells grown on solid agar or colony hybridization wasas easy as for bacterial cells. Expression plasmids carryingyeast-specific promoter (and terminator) sequences could

be used to express foreign genes in the yeast system andeven to design them for export. Remarkably, this approachalso proved that in a multitude of cases human genes werecapable of functionally complementing their homologouscounterparts in appropriate yeast mutants.

� A suitable extension of yeast transformation by plas-mids was offered by the finding that appropriate cosmidsof considerable length (up to 40 kb) could serve as shut-tle vectors as well. This technique was later applied forthe construction of ordered yeast genomic libraries,which turned out to be much more advantageous in thesequencing project than plasmid or phage libraries. Asimilar line was followed in the construction of YACs.Thus, human DNA fragments up to 1 Mb could beaccommodated and propagated. The big hope of usingthis tool in mapping the human genome, however,finally turned into a disappointment because the YACssuffered rearrangements in the yeast due to its propen-sity of frequent recombination via short homologyregions. This trait has been employed in “one-step genereplacement” of yeast genes as only 20 bp at each borderwere sufficient to effect disruption and subsequent sub-stitution of a genomic sequence by another.

� A most successful technique, developed in 1989 and stillapplied, is the yeast two-hybrid system for the detection ofprotein–protein interactions. The yeast three-hybrid sys-tem, developed some 10 years later, was designed as anassay for RNA–protein interactions.

� Finally, we briefly introduce some aspects of techniquesto detect and quantitate protein–protein interactions.

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Further Reading

Chen, R. and Snyder, M. (2010) Yeast proteomics and proteinmicroarrays. Journal of Proteomics, 73, 2147–2157.

Fields, S. and Song, O.K. (1989) A novel genetic system to detectprotein–protein interactions.Nature, 340, 245–246.

Forget, B.G. (1993) YAC transgenes: Bigger is probably better.Proceedings of the National Academy of Sciences of the UnitedStates of America, 90, 7909–7911.

Gietz, D., St Jean, A., Woods, R.A., and Schiestl, R.H. (1992)Improved method for high efficiency transformation ofintact yeast cells. Nucleic Acids Research, 20, 1425.

Kaake, R.M., Wang, X., and Huang, L. (2010) Profiling of pro-tein interaction networks of protein complexes using affin-ity purification and quantitative mass spectrometry.Molecular & Cellular Proteomics, 9, 1650–1665.

Kazuki, Y. and Oshimura, M. (2011) Human artificial chromo-somes for gene delivery and the development of animal

models. Molecular Therapy: The Journal of the American Soci-ety of Gene Therapy, 19, 1591–1601.

SenGupta, D.J., Wickens, M., and Fields, S. (1999) Identifica-tion of RNAs that bind to a specific protein using the yeastthree-hybrid system. RNA (New York, NY), 5, 596–601.

Tsien, R.Y. (2008) Constructing and exploiting the fluorescent pro-tein paintbox. Nobel Lecture, December 8, 2008 (http://www.nobelprize.org/nobel_prizes/chemistry/laureates/2008/).

Volkert, F.C., Wilson, D.W., and Broach, J.R. (1989) Desoxyri-bonucleic acid plasmids in yeast.Microbiological Reviews, 53,299–317.

Wurster, S.E. and Mahler, L.J. III (2010) Selections that opti-mize RNA display in the yeast three-hybrid system. RNA(New York, NY), 16, 253–258.

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