Fred Sherman

An Introduction to the Genetics and Molecular Biology of the Yeast Saccharomyces cerevisiae. (2001) http://dbb.urmc.rochester.edu/labs/sherman_f/yeast/index.html

Table of Contents

• 1 Yeast as a Model Eukaryote • 11 Manipulating the Genome In • 2 Information on Yeast Vitro with Plasmids • 3 Yeast Strains 11.1 Cloning by Complementation • 4 Growth and Life Cycles 11.2 Mutagenesis In Vitro • 5 The Yeast Genome 11.3 Two-step Gene Replacement • 6 Genetic Nomenclature 11.4 Gene Disruption and One-step 6.1 Chromosomal Genes Gene Replacement 6.2 Mitochondrial Gene 11.5 Plasmid Shuffle 6.3 Non-Mendelian Determinants 11.6 Recovering mutant alleles • 7 Genetic Analyses • 12 Interactions of Genes 7.1 Overviews with Examples 12.1 Heterozygosity and Dominant- 7.2 Tetrad Analysis negative Mutations 7.3 Non-Mendelian Inheritance 12.2 Intragenic Complementation • 8 Transformation 12.3 Nonallelic Non- 8.1 Yeast Vector and DNA complementation Fragments 12.4 Suppressors 8.2 Synthetic Oligonucleotides 12.5 Synthetic Enhancement and 8.3 Mitochondrial Transformation Epistatic Relationships • 9 Yeast Vectors • 13 Genomic Analysis 9.1 YIp Vectors • 14 Analyses with Yeast Systems 9.2 YEp Vectors 14.1 Two-hybrid Systems 9.3 YCp Vectors 14.2 Yeast Artificial Chromosomes • 10 Genes Important for Genetic (YACs) Studies 14.3 Expression of Heterologous 10.1 URA3 and LYS2 Protein in Yeast 10.2 ADE1 and ADE2 • Key Words 10.3 GAL1 Promoter • Bibliography 10.4 lacZ and Other Reporters

1 1 Yeast is a Model Eukaryote mutations can be conveniently isolated and manifested in haploid strains, and This chapter deals only with the yeast S. complementation tests can be carried out in cerevisiae, and related interbreeding diploid strains. The development of DNA species. The fission yeast transformation has made yeast particularly Schizosaccharomyces pombe, which is accessible to gene cloning and genetic only distantly related to S. cerevisiae, has engineering techniques. Structural genes equally important features, but is not as corresponding to virtually any genetic trait well characterized. The general principles can be identified by complementation from of the numerous classical and modern plasmid libraries. Plasmids can be approaches for investigating S. cerevisiae introduced into yeast cells either as are described, and the explanation of terms replicating molecules or by integration into and nomenclature used in current yeast the genome. In contrast to most other studies are emphasized . This article should organisms, integrative recombination of be particularly useful to the uninitiated transforming DNA in yeast proceeds who are exposed for the first time to exclusively via homologous experimental studies of yeast. Detailed recombination. Exogenous DNA with at protocols are described in the primary least partial homologous segments can literature and in a number of reviews in the therefore be directed at will to specific books listed in the Bibliography. The locations in the genome. Also, homologous original citations for the material covered recombination, coupled with yeasts’ high in this chapter also can be found in these levels of gene conversion, has led to the comprehensive reviews. development of techniques for the direct replacement of genetically engineered Although yeasts have greater genetic DNA sequences into their normal complexity than bacteria, containing 3.5 chromosome locations. Thus, normal wild- times more DNA than Escherichia coli type genes, even those having no cells, they share many of the technical previously known mutations, can be advantages that permitted rapid progress in conveniently replaced with altered and the molecular genetics of prokaryotes and disrupted alleles. The phenotypes arising their viruses. Some of the properties that after disruption of yeast genes has make yeast particularly suitable for contributed significantly toward biological studies include rapid growth, understanding of the function of certain dispersed cells, the ease of replica plating proteins in vivo. Many investigators have and mutant isolation, a well-defined been shocked to find viable mutants with genetic system, and most important, a little of no detrimental phenotypes after highly versatile DNA transformation disrupting genes that were previously system. Unlike many other assumed to be essential. Also unique to microorganisms, S. cerevisiae is viable yeast, transformation can be carried out with numerous markers. Being directly with synthetic oligonucleotides, nonpathogenic, yeast can be handled with permitting the convenient productions of little precautions. Large quantities of numerous altered forms of proteins. These normal bakers’ yeast are commercially techniques have been extensively exploited available and can provide a cheap source in the analysis of gene regulation, for biochemical studies. structure-function relationships of proteins, chromosome structure, and other general Unlike most other microorganisms, strains questions in cell biology. The overriding of S. cerevisiae have both a stable haploid virtues of yeast are illustrated by the fact and diploid state. Thus, recessive that mammalian genes are being

2 introduced into yeast for systematic Jones et al., 1992; Pringle et al.,1997; analyses of the functions of the Wheals et al., 1995), including protocols corresponding gene products. applicable to yeasts (Fields & Johnson, 1993) and introductory material (Walker, In addition, yeast has proved to be valuable 1998). A more comprehensive listing of for studies of other organisms, including earlier reviews can be found in Sherman the use of the two-hybrid screening system (1991). Interesting and amusing accounts for the general detection of protein-protein of developments in the field are covered in interactions, the use of YACs for cloning The Early Days of Yeast Genetics (Hall & large fragments of DNA, and expression Linder, 1992). The journal Yeast publishes systems for the laboratory and commercial original research articles, reviews, short preparation of heterologous proteins. Many communications, sequencing reports, and of these techniques are described herein. selective lists of current articles on all aspects of Saccharomyces and other yeast During the last two decades, an ever- genera. increasing number of molecular biologists have taken up yeast as their primary Current and frequently-updated research system, resulting in a virtually information and databases on yeast can be autocatalytic stimulus for continuing conveniently retrieved on the Internet investigations of all aspects of molecular through World Wide Web, including the and cell biology. Most significantly, a "Saccharomyces Genomic Information knowledge of the DNA sequence of the Resource" (http://genome- complete genome, which was completed in www.stanford.edu/Saccharomyces/) and 1996, has altered the way molecular and linked files containing DNA sequences, cell biologist approach and carry out their lists of genes, home pages of yeast studies (see Dujon, 1996; Goffeau et al., workers, and other useful information 1996). In addition, plans are under way to concerning yeast. From the MIPS page systematically investigate the possible (http://www.mips.biochem.mpg.de/) you functions of all yeast genes by examining can access the annotated sequence the phenotypes of strains having disrupted information of the genome of genes. Saccharomyces cerevisiae and view the chromosomes graphically or as text, and 2 Information on Yeast more. The YPD page (http://www.proteome.com/YPDhome.htm A general introduction to a few selected l) contains a protein database with topics on yeast can be found in the book emphasis on the physical and functional chapters "Yeast as the E. coli of Eucaryotic properties of the yeast proteins. Cells" and "Recombinant DNA at Work" (Watson et al., 1987). Comprehensive and 3 Yeast Strains excellent reviews of the genetics and molecular biology of S. cerevisiae are Although genetic analyses and contained in three volumes entitled transformation can be performed with a "Molecular Biology of the Yeast number of taxonomically distinct varieties Saccharomyces" (Broach et al., 1991; of yeast, extensive studies have been Jones et al., 1992; Pringle et al., 1997). An limited primarily to the many freely important source for methods used in interbreeding species of the budding yeast genetics and molecular biology of yeast is Saccharomyces and to the fission yeast contained in the book edited by Guthrie Schizosaccharomyces pombe. Although and Fink (1991). Overviews of numerous "Saccharomyces cerevisiae" is commonly subjects are also covered in other sources used to designate many of the laboratory (Broach et al., 1991; Brown & Tuite, 1998; stocks of Saccharomyces used throughout

3 the world, it should be pointed out that laboratory strains produce high frequencies most of these strains originated from the of ρ - mutants. Another strain, D273–10B, interbred stocks of Winge, Lindegren, and has been extensively used as a typical others who employed fermentation normal yeast, especially for mitochondrial markers not only from S. cerevisiae but studies. One should examine the specific also from S. bayanus, S. carlsbergensis, S. characters of interest before initiating a chevalieri, S. chodati, S. diastaticus, etc. study with any strain. Also, there can be a Nevertheless, it is still recommended that high degree of inviability of the meiotic the interbreeding laboratory stocks of progeny from crosses among these Saccharomyces be denoted as S. "normal" strains. cerevisiae, in order to conveniently distinguish them from the more distantly Many strains containing characterized related species of Saccharomyces. auxotrophic, temperature-sensitive, and other markers can be obtained from the Care should be taken in choosing strains Yeast Genetics Stock Culture Center of the for genetic and biochemical studies. American Type Culture Collection Unfortunately there are no truly wild-type (http://www.atcc.org/SearchCatalogs/Yeast Saccharomyces strains that are commonly GeneticStock.cfm), including an almost employed in genetic studies. Also, most complete set of deletion strains domesticated strains of brewers’ yeast and (http://www-deletion.stanford.edu/cgi- probably many strains of bakers’ yeast and bin/deletion/search3.pl.atcc). Currently this true wild-type strains of S. cerevisiae are set consists of 20,382 strains representing not genetically compatible with laboratory deletants of nearly all nonessential ORFs stocks. It is often not appreciated that in different genetic backgrounds. Deletion many "normal" laboratory strains contain strains are also availabe from mutant characters. This condition arose EUROSCARF (http://www.uni- because these laboratory strains were frankfurt.de/fb15/mikro/euroscarf/col_inde derived from pedigrees involving x.html) and Research Genetics mutagenized strains, or strains that carry (http://www.resgen.com/products/YEAST genetic markers. Many current genetic D.php3). Other sources of yeast strains studies are carried out with one or another include the National Collection of Yeast of the following strains or their derivatives, Cultures and these strains have different properties (http://www.ncyc.co.uk/Sacchgen.html) that can greatly influence experimental and the Centraalbureau voor outcomes: S288C; W303; D273–10B; Schimmelcultures X2180; A364A; Σ1278B; AB972; SK1; (http://www2.cbs.knaw.nl/yeast/webc.asp). and FL100. The haploid strain S288C Before using strains obtained from these (MATα SUC2 mal mel gal2 CUP1 flo1 sources or from any investigator, it is flo8-1 hap1) is often used as a normal advisable to test the strains and verify their standard because the sequence of its genotypes. genome has been determined (Goffeau et al., 1996), because many isogenic mutant 4 Growth and Life Cycles derivatives are available, and because it gives rise to well-dispersed cells. However, Vegetative cell division of yeast S288C contains a defective HAP1 gene, characteristically occurs by budding, in making it incompatible with studies of which a daughter is initiated as an out mitochondrial and related systems. Also, in growth from the mother cell, followed by contrast to Σ1278B, S288C does not form nuclear division, cell-wall formation, and pseudohyae. While true wild-type and finally cell separation. The sizes of haploid domesticated bakers’ yeast give rise to less and diploid cells vary with the phase of than 2% ρ - colonies (see below), many growth and from strain to strain. Typically,

4 diploid cells are 5 x 6 µm ellipsoids and In addition, certain diploid strains of S. haploid cells are 4 µm diameter spheroids. cerevisiae can assume a markedly different The volumes and gross composition of cell and colony morphology, denoted yeast cells are listed in Table 1. During pseudohyphae, when grown on agar exponential growth, haploid cultures tend medium limiting for nitrogen sources. to have higher numbers of cells per cluster These pseudohyphal cells are significantly compared to diploid cultures. Also haploid elongated, and mother-daughter pairs cells have buds that appear adjacent to the remain attached to each other. This previous one; whereas diploid cells have characteristic pseudohyphal growth causes buds that appear at the opposite pole. Each extended growth of branched chains mother cell usually forms no more than 20- outward from the center of the colony, and 30 buds, and it age can be determined by invasive growth under the surface of agar the number of bud scars left on the cell medium. wall. Table 4.1. Size and composition of yeast cells

Characteristic Haploid cell Diploid cell

Volume (µm3) 70 120 Composition (10-12 g) Wet weight 60 80 Dry weight 15 20 DNA 0.017 0.034 RNA 1.2 1.9 Protein 6 8

"Normal" laboratory haploid strains have a heterothallic and homothallic diploid doubling time of approximately 90 min. in strains sporulate under conditions of complete YPD (1% yeast extract, 2% nutrient deficiency, and especially in peptone, and 2% glucose) medium and special media, such as potassium acetate approximately 140 min. in synthetic media medium. During sporulation, the diploid during the exponential phase of growth at cell undergoes meiosis yielding four the optimum temperature of 30°C. progeny haploid cells, which become However, strains with greatly reduced encapsulated as spores (or ascospores) growth rates in synthetic media are often within a sac-like structure called an ascus encountered. Usually strains reach a (plural asci). The percent sporulation maximum density of 2 x108 cells/ml in varies with the particular strain, ranging YPD medium. Titers 10 times this value from no or little sporulation to nearly can be achieved with special conditions, 100%. Many laboratory strains sporulate to such as pH control, continuous additions of over 50%. The majority of asci contains balanced nutrients, filtered-sterilized media four haploid ascospores, although varying and extreme aeration that can be delivered proportions asci with three or less spores in fermenters. are also observed.

S. cerevisiae can be stably maintained as either heterothallic or homothallic strains, as illustrated in Figure 4.1. Both

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Figure 4.1. Life cycles of heterothallic and homothallic strains of S. cerevisiae. Heterothallic strains can be stably maintained as diploids and haploids, whereas homothallic strains are stable only as diploids, because the transient haploid cells switch their mating type, and mate.

Because the a and α mating types are diploid strain cannot be selected, zygotes under control of a pair of MATa/MATα can be separated from the mating mixture heterozygous alleles, each ascus contains with a micromanipulator. Zygotes are two MATa and two MATα haploid cells. identified by a characteristic thick zygotic Upon exposure to nutrient condition, the neck, and are best isolated 4 to 6 hr after spores germinate, vegetative growth incubating the mixture when the mating commences and mating of the MATa and process has just been completed. MATα can occur. However, if the haploid spores are mechanically separated by 5 The Yeast Genome micromanipulation, the haplophase of heterothallic strains can be stably S. cerevisiae contains a haploid set of 16 maintained, thus allowing the preparation well-characterized chromosomes, ranging of haploid strains. In contrast, the presence in size from 200 to 2,200 kb. The total of the HO allele in homothallic strains sequence of chromosomal DNA, causes switching of the mating type in constituting 12,052 kb, was released in growing haploid cells, such that MATa April, 1996. A total of 6,183 open-reading- cells produce MATα buds and MATα cells frames (ORF) of over 100 amino acids produce MATa buds. As a consequence, long were reported, and approximately mating occurs and there is only a transient 5,800 of them were predicated to haplophase in homothallic strains (Figure correspond to actual protein-coding genes. 4.1). A larger number of ORFs were predicted by considering shorter proteins. In contrast Controlled crosses of MATa and MATα to the genomes of multicellular organsims, haploid strains are simply carried out by the yeast genome is highly compact, with mixing approximately equal amounts of genes representing 72% of the total each strain on a complete medium and sequence. The average size of yeast genes incubating the mixture at 30°C for at least is 1.45 kb, or 483 codons, with a range 6 hr. Prototrophic diploid colonies can then from 40 to 4,910 codons. A total of 3.8% be selected on appropriate synthetic media of the ORF contain introns. Approximately if the haploid strains contain 30% of the genes already have been complementing auxotrophic markers. If the characterized experimentally. Of the

6 remaining 70% with unknown function, translational machinery and approximately approximately one half either contain a 15% of the mitochondrial proteins. ρo motif of a characterized class of proteins or mutants completely lack mitochondrial correspond to genes encoding proteins that DNA and are deficient in the respiratory are structurally related to functionally polypeptides synthesized on mitochondrial characterized gene products from yeast or ribosomes, i.e., cytochrome b and subunits from other organisms. of cytochrome oxidase and ATPase complexes. Even though ρo mutants are Ribosomal RNA is coded by respiratory deficient, they are viable and approximately 120 copies of a single still retain mitochondria, although tandem array on chromosome XII. The morphologically abnormal. DNA sequence revealed that yeast contains 262 tRNA genes, of which 80 have introns. The 2-µm circle plasmids, present in most In addition, chromosomes contain movable strains of S. cerevisiae, apparently function DNA elements, retrotransposons, that vary solely for their own replication. Generally in number and position in different strains ciro strains, which lack 2-µm DNA, have of S. cerevisiae, with most laboratory no observable phenotype. However, a strains having approximately 30. certain chromosomal mutation, nib1, causes a reduction in growth of cir+ strains, Other nucleic acid entities, presented in due to an abnormally high copy number 2- Figure 5.1, also can be considered part of µm DNA. the yeast genome. Mitochondrial DNA encodes components of the mitochondrial

Figure 5.1. The genome of a diploid cell of S. cerevisiae (see the text). A wild-type chromosomal gene is depicted as YFG1+ (Your Favorite Gene) and the mutation as yfg1-1.

Similarly, almost all S. cerevisiae strains particles that are transmitted contain dsRNA viruses, that constitutes cytoplasmically during vegetative growth approximately 0.1% of total nucleic acid. and conjugation. L-B and L-C (collectively RNA viruses include three families with denoted L-BC), similar to L-A, have a dsRNA genomes, L-A, L-BC, and M. Two RNA-dependent RNA polymerase and are other families of dsRNA, T and W, present in intracellular particles. KIL-o replicate in yeast but so far have not been mutants, lacking M dsRNA and shown to be viral. M dsRNA encodes a consequently the killer toxin, are readily toxin, and L-A encodes the major coat induced by growth at elevated protein and components required for the temperatures, and chemical and physical viral replication and maintenance of M. agents. The two dsRNA, M and L-A, are packaged separately with the common capsid protein Yeast also contains a 20S circular single- encoded by L-A, resulting in virus-like stranded RNA (not shown in Figure 5.1)

7 that appears to encode an RNA-dependent tetrads after sporulation of heterozygous RNA polymerase, that acts as an diploids; this property is dependent on the independent replicon, and that is inherited disjunction of chromosomal centromeres. as a non-Mendelian genetic element. In contrast, non-Mendelian inheritance is observed for the phenotypes associated Only mutations of chromosomal genes with the absence or alteration of other exhibit Mendelian 2:2 segregation in nucleic acids described in Figure 5.1.

6 Genetic Nomenclature

6.1 Chromosomal Genes

Table 6.1. Genetic nomenclature, using ARG2 as an example

Gene Definition symbol

ARG+ All wild-type alleles controlling arginine requirement ARG2 A locus or dominant allele arg2 A locus or recessive allele confering an arginine requirement arg2- Any arg2 allele confering an arginine requirement ARG2+ The wild-type allele arg2-9 A specific allele or mutation Arg+ A strain not requiring arginine Arg- A strain requiring arginine Arg2p The protein encoded by ARG2 Arg2 protein The protein encoded by ARG2 ARG2 mRNA The mRNA transcribed from ARG2 arg2-∆1 A specific complete or partial deletion of ARG2 ARG2::LEU2 Insertion of the functional LEU2 gene at the ARG2 locus, and ARG2 remains functional and dominant arg2::LEU2 Insertion of the functional LEU2 gene at the ARG2 locus, and arg2 is or became nonfunctional arg2- Insertion of the functional LEU2 gene at the ARG2 locus, and the specified 10::LEU2 arg2-10 allele which is nonfunctional cyc1-arg2 A fusion between the CYC1 and ARG2 genes, where both are nonfunctional PCYC1-ARG2 A fusion between the CYC1 promoter and ARG2, where the ARG2 gene is functional

8 The genetic nomenclature for cyc1-717/CYC1+ cross and dominant in the chromosomal genes of the yeast S. CYC1-717/cyc1-∆1 cross. Thus, sometimes cerevisiae is now more-or-less universally it is less confusing to denote all mutant accepted, as illustrated in Table 6.1, using alleles in lower case letters, especially ARG2 as an example. Whenever possible, when considering a series of mutations each gene, allele, or locus is designated by having a range of activities. three italicized letters, e.g., ARG, which is usually a describer, followed by a number, Although superscript letters should be e.g., ARG2. Unlike most other systems of avoided, it is sometimes expedient to genetic nomenclature, dominant alleles are distinguish genes conferring resistance and denoted by using uppercase italics for all sensitivity by superscript R and S, letters of the gene symbol, e.g., ARG2, respectively. For example, the genes whereas lowercase letters denote the controlling resistance to canavanine recessive allele, e.g., the auxotrophic sulphate (can1) and copper sulphate marker arg2. Wild-type genes are (CUP1) and their sensitive alleles could be designated with a superscript "plus" (sup6+ R R + denoted, respectively, as can 1, CUP 1, or ARG2 ). Alleles are designated by a CANS1, and cupS1. number separated from the locus number by a hyphen, e.g., arg2-9. The symbol ∆ Wild-type and mutant alleles of the can denote complete or partial deletions, mating-type locus and related loci do not e.g., arg2-∆1. Insertion of genes follow the follow the standard rules. The two wild- bacterial nomenclature by using the type alleles of the mating-type locus are symbol :: . For example, arg2::LEU2 designated MATa and MATα. The wild- denotes the insertion of the LEU2 gene at type homothallic alleles at the HMR and the ARG2 locus, in which LEU2 is HML loci are denoted, HMRa, HMRα, dominant (and functional), and arg2 is HMLa and HMLα. The mating phenotypes recessive (and defective). of MATa and MATα cells are denoted simply a and α, respectively. The two Phenotypes are sometimes denoted by letters HO denotes the gene encoding the cognate symbols in roman type and by the endonuclease required for homothallic superscripts + and -. For example, the switching. independence and requirement for arginine can be denoted by Arg+ and Arg-, Dominant and recessive suppressors respectively. Proteins encoded by ARG2, should be denoted, respectively, by three for example, can be denoted Arg2p, or uppercase or three lowercase letters, simply Arg2 protein. However, gene followed by a locus designation, e.g., symbols are generally used as adjectives SUP4, SUF1, sup35, suf11, etc. In some for other nouns, for example, ARG2 instances UAA ochre suppressors and mRNA, ARG2 strains, etc. UAG amber suppressors are further designated, respectively, o and a following Although most alleles can be the locus. For example, SUP4-o refers to unambiguously assigned as dominant or suppressors of the SUP4 locus that insert recessive by examining the phenotype of tyrosine residues at UAA sites; SUP4-a the heterozygous diploid crosses, dominant refers to suppressors of the same SUP4 and recessive traits are defined only with locus that insert tyrosine residues at UAG pairs, and a single allele can be both sites. The corresponding wild-type locus dominant and recessive. For example, that encodes the normal tyrosine tRNA and because the alleles CYC1+, cyc1-717 and that lacks suppressor activity can be cyc1-∆1 produce, respectively, 100%, 5% referred to as sup4+. Intragenic mutations and 0% of the gene product, the cyc1-717 that inactivate suppressors can denoted, for allele can be considered recessive in the example, sup4- or sup4-o-1. Frameshift 9 suppressors are denoted as suf (or SUF), 6.2 Mitochondrial Genes whereas metabolic suppressors are denoted with a variety of specialized symbols, such Special consideration should be made of as ssn (suppressor of snf1), srn (suppressor the nomenclature describing mutations of of rna1-1), and suh (suppressor of his2-1) mitochondrial components and function that are determined by both nuclear and Capital letters are also used to designate mitochondrial DNA genes. The growth on certain DNA segments whose locations media containing nonfermentable have been determined by a combination of substrates (Nfs) as the sole energy and recombinant DNA techniques and classical carbon source (such as glycerol or ethanol) mapping procedures, e.g., RDN1, the is the most convenient operational segment encoding ribosomal RNA. procedure for testing mitochondrial function. Lack of growth on The general form YCRXXw is now used to nonfermentable media (Nfs- mutants), as designate genes uncovered by well as other mitochondrial alterations, can systematically sequencing the yeast be due to either nuclear or mitochondrial genome, where Y designates yeast; C (or mutations as outlined in Table 3. Nfs- A, B, etc.) designates the chromosome III nuclear mutations are generally denote by (or I, II, etc.); R (or L) designates the right the symbol pet; however, more specific (or left) arm of the chromosome; XX designations have been used instead of pet designates the relative position of the start when the gene products were known, such of the open-reading frame from the as cox4, hem1, etc. centromere; and w (or c) designates the Watson (or Crick) strand. For example, The complexity of nomenclatures for YCR5c denotes CIT2, a previously known mitochondrial DNA genes, outlined in but unmapped gene situated on the right Table 6.2, is due in part to complexity of arm of chromosome III, fifth open reading- the system, polymorphic differences of frame from the centromere on the Crick mitochondrial DNA, complementation strand. between exon and intron mutations, the presence of intron-encoded maturases, E. coli genes inserted into yeast are usually diversed phenotypes of mutations within denoted by the prokaryotic nomenclature, the same gene, and the lack of agreement e. g., lacZ. between various workers. Unfortunately, the nomenclature for most mitochondrial A list of gene symbols are tabulated in the mutations do not follow the rules outline book edited by Wheals et al. (1995), for nuclear mutations. Furthermore, whereas a current list can be found in the confusion can occur between phenotypic Internet file designations, mutant isolation number, ftp://genome- allelic designations, loci, and cistrons ftp.stanford.edu/pub/yeast/gene_registry/re (complementation groups). gistry.genenames.tab

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Table 6.2 Mitochondrial genes and mutations with examples

Wild- Mutation Mutant phenotype or gene product type (with examples)

Nuclear genes PET+ pet- Nfs- pet1 Unknown function cox4 Cytochrome c oxidase subunit IV hem1 δ-Aminolevulinate synthase cyc3 Cytochrome c heme lyase Mitochondrial DNA Gross aberrations ρ+ ρ- Nfs- ρo ρ- mutants lacking mitochondrial DNA Single-site mutations ρ+ mit- Nfs-, but capable of mitochondrial translation [COX1] [cox1] Cytochrome c oxidase subunit I [COX2] [cox2] Cytochrome c oxidase subunit II [COX3] [cox3] Cytochrome c oxidase subunit III [COB1] [cob1] or [box] Cytochrome b [ATP6] [atp6] ATPase subunit 6 [ATP8] [atp8] ATPase subunit 8 [ATP9] [atp9] or [pho2] ATPase subunit 9 [VAR1] Mitochondrial ribosomal subunit ρ+ syn- Nfs-, deficient in mitochondrial translation tRNAAsp or M7-37 Mitochondrial tRNAAsp (CUG) R ant Resistant to inhibitors S R [ery ] ery or [rib1] Resistant to erythromycin, 21S rRNA S R [cap ] cap or [rib3] Resistant to chloramphenical, 21S rRNA S R [par ] par or [par1] Resistant to paromomycin, 16S rRNA S R [oli ] oli or [oli1] Resistant to oligomycin, ATPase subunit 9

Nfs- denotes lack of growth on nonfermentable substrates.

11 6.3 Non-Mendelian Determinants

In addition to the non-Mendelian determinants described in Figure 5.1 (2 µm plasmid, mitochondrial genes, and RNA viruses) and discussed in Section 5 (The Yeast Genome), yeast contains elements that have been proposed to be prions, i.e., infectious proteins, on the bases of their genetic properties. The nomenclature of these putative prions, representing alternative protein states, are presented in Table 6.4.

Table 6.4. Nomenclature of presumptive prions exhibiting non-Mendelian inhertance

Putative Prion state gene Positive Negative product Phenotype of negative state

ψ+ ψ - Sup35p Decreased efficiency of certain suppression ξ + ξ - Sup35p Decreased efficiency of certain suppression [URE3] [ure3-] Ure2p Deficiency in ureidosuccinate utilization

7 Genetic Analyses previously defined mutations causing the same phenotype. The diploid crosses are 7.1 Overviews with Examples isolated and the Yfg trait is scored. The Yfg+ phenotype in the heterozygous - There are numerous approaches for the control cross establishes that the Yfg - isolation and characterization of mutations mutation is recessive. The Yfg phenotype in yeast. Generally, a haploid strain is in MATα yfg1 cross, and the Yfg+ treated with a mutagen, such as phenotype in the MATα yfg2, MATα yfg3, ethylmethanesulfonate, and the desired etc., crosses reveals that the original Yfg- mutants are detected by any one of a mutant contains a yfg1 mutation. number of procedures. For example, if Yfg- (Your Favorite Gene) represents an Meiotic analysis can be used to determine auxotrophic requirement, such as arginine, if a mutation is an alteration at a single or temperature-sensitive mutants unable to genetic locus and to determine genetic grow at 37°C, the mutants could be scored linkage of the mutation both to its by replica plating. Once identified, the centromere and to other markers in the Yfg- mutants could be analyzed by a cross. As illustrated in Figure 7.1, the variety of genetic and molecular methods. MATa yfg1 mutant is crossed to a normal Three major methods, complementation, MATα strain. The diploid is isolated and meiotic analysis and molecular cloning are sporulated. Typically, sporulated cultures illustrated in Figure 7.1. contain the desired asci with four spores, as well as unsporulated diploid cells and Genetic complementation is carried out by rare asci with less than four spores. The crossing the Yfg- MATa mutant to each of sporulated culture is treated with snail the tester strains MATα yfg1, MATα yfg2, extract which contains an enzyme that etc., as well as the normal control strain dissolves the ascus sac, but leaves the four MATα. These yfg1, yfg2, etc., are spores of each tetrad adhering to each

12 other. A portion of the treated sporulated the Yfg+:Yfg- phenotypes is indicative of a culture is gently transferred to the surface single gene. If other markers are present in of a petri plate or an agar slab. The four the cross, genetic linkage of the yfg1 spores of each cluster are separated with a mutation to the other markers or to the microneedle controlled by a centromere of its chromosome could be micromanipulator. After separation of the revealed from the segregation patterns. desired number of tetrads, the ascospores are allowed to germinate and form colonies The molecular characterization of the yfg1 on complete medium. The haploid mutation can be carried out by cloning the segregants can then be scored for the Yfg+ wild-type YFG1+ gene by and Yfg- phenotypes. Because the four complementation, as illustrated in Figure spores from each tetrad are the product of a 7.1 and described below (Section 11.1 single meiotic event, a 2:2 segregation of Cloning by Complementation).

Figure 7.1. General approaches for genetic analysis. As an example, a MATa strain is mutagenized and a hypothetical trait, Yfg- (Your Favorite Gene) is detected. The Yfg- mutant is analyzed by three methods, complementation, meiotic analysis and molecular cloning (see the text).

7.2 Tetrad analysis event, and the genetic analysis of these tetrads provides a sensitive means for Meiotic analysis is the traditional method determining linkage relationships of genes for genetically determining the order and present in the heterozygous condition. It is distances between genes of organisms also possible to map a gene relative to its having well-defined genetics systems. centromere if known centromere-linked Yeast is especially suited for meiotic genes are present in the cross. Although mapping because the four spores in an the isolation of the four spores from an ascus are the products of a single meiotic ascus is one of the more difficult

13 techniques in yeast genetics, requiring a analysis is necessary for determining a micromanipulator and practice, tetrad mutation corresponds to an alteration at a analysis is routinely carried out in most single locus, for constructing strains with laboratories working primarily with yeast. new arrays of markers, and for Even though linkage relationships are no investigating the interaction of genes. longer required for most studies, tetrad

Figure 7.2. Origin of different tetrad types. Different tetrad types (left) are produced with genes on homologous (center) or nonhomologous (right) chromosomes from the cross AB x ab. When PD > NPD, then the genes are on homologous chromosomes, because of the rarity of NPD, which arise from four strand double crossovers. The tetratype (T) tetrads arise from single crossovers. See the text for the method of converting the %T and %NPD tetrads to map distances when genes are on homologous chromosomes. If gene are on nonhomologous chromosomes, or if they greatly separated on the same chromosome, then PD = NPD, because of independent assortment, or multiple crossovers. Tetratype tetrads of genes on nonhomologous chromosomes arise by crossovers between either of the genes and their centromere, as shown in the lower right of the figure. The %T can be used to determine centromere distances if it is known for one of the genes (see the text).

14 There are three classes of tetrads from a hybrid which is heterozygous for two markers, AB x ab: PD (parental ditype), NPD (non-parental ditype) and T (tetratype) as shown in Figure 7.2. The following ratios of these tetrads can be used to deduce gene and centromere linkage:

PD NPD T

AB aB AB AB aB Ab ab Ab ab ab Ab aB

Random assortment 1 : 1 : 4 Linkage >1 : <1 Centromere linkage 1 : 1 : <4

There is an excess of PD to NPD asci if two genes are linked. If two genes are on The equation for deducing map distances, different chromosomes and are linked to cM, is accurate for distances up to their respective centromeres, there is a approximately 35 cM. For larger distances reduction of the proportion of T asci. If up to approximately 75 cM, the value can two genes are on different chromosomes be corrected by the following empirically- and at least one gene is not centromere- derived equation: linked, or if two genes are widely separated on the same chromosome, there is independent assortment and the PD : NPD : T ratio is 1 : 1 : 4. The origin of different tetrad types are illustrated in Figure 7.2. Similarly, the distance between a marker and its centromere cM', can be The frequencies of PD, NPD, and T tetrads approximated from the percentage of T can be used to determine the map distance tetrads with a tightly-linked centromere in cM (centimorgans) between two genes if marker, such as trp1: there are two or lesser exchanges within the interval:

______

7.3 Non-Mendelian Inheritance Each ascus from a ρ+ diploid strain contains four ρ+ segregants or a ratio of 4:0 The inheritance of non-Mendelian for ρ+:ρ-. In contrast, a cross of pet1 MATa elements can be revealed by tetrad and PET1+ MATα strains would result in a + analysis. For example, a cross of ρ MATa PET1+/pet1 MATα/MAT diploid, which - a and ρ MATα haploid strains would result would yield a 2:2 segregation of + - in ρ MATa/MATα and ρ MATa/MΑΤα PET1+/pet1. Similar, the other non- diploid strains, the proportion of which Mendelian determinants also produce would depend on the particular ρ- strain.

15 primarily 4:0 or 0:4 segregations after the use of shuttle vectors which can be meiosis. used to transform both yeast and E. coli.

Another means for analyzing non- The following three main methods are Mendelian elements is cytoduction, which currently used to transform yeast: (i) those is based on the segregation of haploid using spheroplasts; or (ii) cells treated with cells, either MATa or MATα, from zygotes. lithium salts; and (iii) the use of Haploid cells arise from zygotes at electroporation. frequencies of approximately 10-3 with normal strains, and nearly 80% with kar1 Spheroplasts for transformations are crosses, such as, for example, kar1 MATa prepared by the action of hydrolytic x KAR1+ MATα. While the haploid enzymes to remove portions of the cell segregants from a kar1 cross generally wall in the presence of osmotic stabilizers, retains all of the chromosomal markers typically 1 M sorbitol. Cell-wall digestion from either the MATa or MATα parental is carried out either with a snail-gut strain, the non-Mendelian elements can be extract, usually denoted Glusulase, or with reassorted. For example, a MATa canR1 Zymolyase, an enzyme from Arthrobacter kar1 [ρ- ψ- kil-o] x MATα CANS1 [ρ+ ψ+ luteus. DNA is added to the spheroplasts, kil-k] cross can yield MATa canR1 kar1 and the mixtures is co-precipitated with a + + solution of polyethylene glycol (PEG) and haploid segregants that are [ρ ψ kil-k], 2+ [ρ- ψ+ kil-k], etc. In addition, high Ca . Subsequently, the cells are resuspended in a solution of sorbitol, frequencies of 2 µm plasmids and low mixed with molten agar and then layered frequencies of chromosome can leak from on the surface of a selective plate one nucleus to another. containing sorbitol. Although this protocol is particularly tedious, and efficiency of Also, the mating of two cells with different transformation can vary by over four mitochondrial DNAs results in a orders of magnitude with different strains, heteroplasmic zygote containing both very high frequencies of transformation, mitochondrial genomes. Mitotic growth of 4 the zygote usually is accompanied by rapid over 10 transformants/µg DNA, can be segregation of homoplasmic cells obtained with certain strains. containing either one of the parental mitochondrial DNAs or a recombinant Most investigators use cells treated with product. The frequent recombination and lithium salts for transformation. After rapid mitotic segregation of mitochondrial treating the cells with lithium acetate, DNAs can be seen, for example, by mating which apparently permeabilizes the cell two different mit- strains, and observing wall, DNA is added and the cells are co- both Nfs- parental types as well as the Nfs+ precipitated with PEG. The cells are recombinant (see Table 6.2). exposed to a brief heat shock, washed free of PEG and lithium acetate, and subsequently spread on plates containing

8 Transformation ordinary selective medium. Increased frequencies of transformation are obtained

8.1 Yeast Vector and DNA Fragments by using specially-prepared single-stranded carrier DNA and certain organic solvents. In general, transformation is the introduction into cells of exogenously A commonly-used method for added DNA and the subsequent inheritance transforming a wide range of different and expression of that DNA. The most species of cells is based on the induced important advances in the molecular permeability to DNA by exposure to characterization and controlled electrical fields. The interaction of an modification of yeast genes have relied on 16 external electric field with the lipid dipoles 8.3 Mitochondrial Transformation of a pore configuration is believed to induce and stabilize the permeation sites, Standard methods for transformation of resulting in cross membrane transport. nuclear genes are ineffective for Freshly-grown yeast cultures are washed, mitochondrial DNA genes. However, DNA suspended in an osmotic protectant, such can be delivered to the mitochondrial as sorbitol, DNA is added, and the cell matrix by high-velocity bombardment of suspension is pulsed in an electroporation yeast cells with tungsten microprojectiles device. Subsequently, the cells are spread carrying mitochondrial DNA. Several on the surface of plates containing high-velocity microprojectile selective media. The efficiency of bombardment devices are commercially transformation by electroporation can be available, and these are powered by increased over 100-fold by using PEG, gunpowder charge or compressed gas. single-stranded carrier DNA and cells that are in late log-phase of growth. Although This method was used to demonstrated that electroporation procedures are simple, the ρo strains can be converted to stable specialized equipment and the required "synthetic ρ-" strains by transformation cuvettes are costly. with bacterial plasmids carrying mitochondrial genes (see Table 6.2). 8.2 Synthetic Oligonucleotides Similar to natural ρ- mitochondrial DNA, the synthetic ρ- mitochondrial DNA can A convenient procedure has been described recombine with ρ+ mitochondrial DNA, for producing specific alterations of chromosomal genes by transforming yeast thus providing means to replace ρ+ wild- in directly with synthetic oligonucleotides. type genes with mutations generated vitro This procedure is easily carried out by . transforming a defective mutant and selecting for at least partially functional Synthetic ρ- strains are isolated by o revertants. Transformation of yeast directly bombarding a lawn of ρ cells on the with synthetic oligonucleotides is thus surface of a petri plate with YEp or YCp ideally suited for producing a large number plasmids carrying both a selectable marker, of specific alterations that change a such as URA3, and the mitochondrial gene completely nonfunctional allele to at least of interest. The nuclear and mitochondrial a partially functional form. The genes may either be on separate or the + oligonucleotide should contain a sequence same plasmid. Ura colonies, for example, that would correct the defect and produce are then screen for the presence of the the desired additional alterations at nearly mitochondrial gene by crossing the - sites. The method is apparently applicable colonies to an appropriate mit tester strain + to all mutant alleles whose functional and scoring the diploids for Nfs (see forms can be selected. Although it is a Table 3). The efficiency of mitochondrial general procedure, so far it has been transformation varies from experiment to -3 extensively used only with mutations of experiment, and can be from 2 x 10 to -4 CYC1, that encodes iso-1-cytochrome c, less than 10 mitochondrial transformants and CYT1 that encodes cytochrome c1. per nuclear transformant. The transformation is carried out by the usual lithium acetate procedure, using 9 Yeast Vectors approximately 50 µg of oligonucleotides that are approximately 40 nucleotides long. A wide range of vectors are available to meet various requirements for insertion, deletion alteration and expression of genes in yeast. Most plasmids used for yeast

17 studies are shuttle vectors, which contain markers include URA3, HIS3, LEU2, TRP1 sequences permitting them to be selected and LYS2, which complement specific and propagated in E. coli, thus allowing for auxotrophic mutations in yeast, such as convenient amplification and subsequent ura3-52, his3-∆1, leu2-∆1, trp1-∆1 and alteration in vitro. The most common yeast lys2-201. These complementable yeast vectors originated from pBR322 and mutations have been chosen because of contain an origin of replication (ori), their low-reversion rate. Also, the URA3, promoting high copy-number maintenance HIS3, LEU2 and TRP1 yeast markers can in E. coli, and the selectable antibiotic complement specific E. coli auxotrophic markers, the β-lactamase gene, bla (or mutations. AmpR), and sometime to tetracycline- resistance gene, tet or (TetR), conferring The URA3 and LYS2 yeast genes have an resistance to, respectively, ampicillin and additional advantage because both positive tetracycline. and negative selections are possible, as discussed below (Section 10.1, URA3 and In addition, all yeast vectors contain LYS2). markers that allow selection of transformants containing the desired plasmid. The most commonly used yeast

Table 9.1. Components of common yeast plasmid vectors

YIp YEp YRp YCp

Plasmid E. coli genes or segments ori, bla; tet + + + + Yeast genes or segments URA3; HIS3; LEU2; TRP1; LYS2; etc. + + + + leu2-d 0 + + 0 2 µm; 2 µm-ori REP3; 0 + 0 0 ARS1; ARS2; ARS3; etc. 0 0 + + CEN3; CEN4; CEN11; etc. 0 0 0 + Host (yeast) markers ura3-52; his3-∆1; leu2-∆1; trp1-∆1; lys2-201; etc. + + + +

Stability ++ + ± +

Although there are numerous kinds of number vectors, YEp; or autonomously yeast shuttle vectors, those used currently replicating low copy-number vectors, YCp. can be broadly classified in either of Another type of vector, YACs, for cloning following three types as summarized in large fragments are discussed in Section Table 9.1: integrative vectors, YIp; 13.2 (Yeast Artificial Chromosomes). autonomously replicating high copy-

18 9.1 YIp Vectors can be used to construct stable strains overexpressing specific genes. YIp The YpI integrative vectors do not plasmids with two yeast segments, such as replicate autonomously, but integrate into YFG1 and URA3 marker, have the the genome at low frequencies by potential to integrate at either of the homologous recombination. Integration of genomic loci, whereas vectors containing circular plasmid DNA by homologous repetitive DNA sequences, such as Ty recombination leads to a copy of the vector elements or rDNA, can integrate at any of sequence flanked by two direct copies of the multiple sites within genome. Strains the yeast sequence as illustrated in the top constructed with YIp plasmids should be of Figure 5. The site of integration can be examined by PCR analysis, or other targeted by cutting the yeast segment in the methods, to confirm the site of integration. YIp plasmid with a restriction endonuclease and transforming the yeast Strains transformed with YIp plasmids are strain with the linearized plasmid. The extremely stable, even in the absence of linear ends are recombinogenic and direct selective pressure. However, plasmid loss integration to the site in the genome that is can occur at approximately 10-3 to 10-4 homologous to these ends. In addition, frequencies by homologous recombination linearization increases the efficiency of between tandemly repeated DNA, leading integrative transformation from 10- to 50- to looping out of the vector sequence and fold. one copy of the duplicated sequence as illustrated in Figure 9.1 and discussed The YIp vectors typically integrate as a below in Section 11.2 (Two-Step Gene single copy. However multiple integration Replacement). do occur at low frequencies, a property that

Figure 9.1. Two-step gene replacement. The wild-type chromosomal YFG1+ allele can be replaced by the mutant yfg1-1 allele from a YIp integrating plasmid. The plasmid is first integrated in the chromosome corresponding to the site on the plasmid that was cleaved by a restriction endonuclease. Strains that have excised the URA3 marker in vivo by homologous recombination are selected on FOA medium. Either the original YFG1+ allele, or the yfg1-1 allele remains in the chromosome, depending on the site of the cross-over.

19

9.2 YEp Vectors vectors are useful in large-scale cultures with complete media where plasmid The YEp yeast episomal plasmid vectors selection is not possible. The most replicate autonomously because of the common use for YEp plasmid vectors is to presence of a segment of the yeast 2 µm overproduce gene products in yeast. plasmid that serves as an origin of replication (2 µm ori). The 2 µm ori is 9.3 YCp Vectors responsible for the high copy-number and high frequency of transformation of YEp The YCp yeast centromere plasmid vectors vectors. are autonomously replicating vectors containing centromere sequences, CEN, YEp vectors contain either a full copy of and autonomously replicating sequences, the 2 µm plasmid, or, as with most of these ARS. The YCp vectors are typically kinds of vectors, a region which present at very low copy numbers, from 1 encompasses the ori and the REP3 gene. to 3 per cell, and possibly more, and are -2 The REP3 gene is required in cis to the ori lost in approximately 10 cells per for mediating the action of the trans-acting generation without selective pressure. In REP1 and REP2 genes which encode many instances, the YCp vectors segregate products that promote partitioning of the to two of the four ascospore from an ascus, plasmid between cells at division. indicating that they mimic the behavior of Therefore, the YEp plasmids containing chromosomes during meiosis, as well as the region encompassing only ori and during mitosis. The ARS sequences are REP3 must be propagated in cir+ hosts believed to correspond to the natural containing the native 2 µm plasmid (Figure replication origins of yeast chromosomes, 5.1). and all of them contain a specific consensus sequence. The CEN function is Most YEp plasmids are relatively unstable, dependent on three conserved domains, being lost in approximately 10-2 or more designated I, II, and III; all three of these cells after each generation. Even under elements are required for mitotic conditions of selective growth, only 60% stabilization of YCp vectors. YRp vectors, to 95% of the cells retain the YEp plasmid. containing ARS but lacking functional CEN elements, transform yeast at high The copy number of most YEp plasmids frequencies, but are lost at too high a ranges from 10-40 per cell of cir+ hosts. frequency, over 10% per generation, However, the plasmids are not equally making them undesirable for general distributed among the cells, and there is a vectors. high variance in the copy number per cell in populations. The stability and low copy-number of YCp vectors make them the ideal choice for Several systems have been developed for cloning vectors, for construction of yeast producing very high copy-numbers of YEp genomic DNA libraries, and for plasmids per cell, including the use of the investigating the function of genes altered partially defective mutation leu2-d, whose in vivo. ARS1, which is in close proximity expression is several orders of magnitude to TRP1, is the most commonly used ARS less than the wild-type LEU2+ allele. The element for YCp vectors, although others copy number per cell of such YEp leu2-d have been used. CEN3, CEN4 and CEN11 vectors range from 200-300, and the high are commonly used centromeres that can copy-number persists for many generations be conveniently manipulated. For example, after growth in leucine-containing media the vector YCp50 contains CEN4 and without selective pressure. The YEp leu2-d ARS1.

20 10 Genes Important for Genetic which is the most commonly used host Studies marker, contains a Ty1 insertion, is not revertible, and does not allow integation of Several genes and promoters are YIp-URA3 plasmids at the URA3 commonly employed for genetic chromosomal locus in most, but not all manipulations and studies with yeast. strains. Some of these genes have special properties, whereas others were originally LYS2 encodes α-aminoadipate reductase, choosen arbitarily and are conveniently an enzyme which is required for the - - available in strains and plasmids. Several biosynthesis of lysine. Lys2 and lys5 of the genes most commonly used for a mutants, but not normal strains grow on a variety of purposes are described below. medium lacking the normal nitrogen source, but containing lysine and αAA. 10.1 URA3 and LYS2 Apparently, lys2 and lys5 mutations cause the accumulation of a toxic intermediate of The URA3 and LYS2 yeast genes have a lysine biosynthesis that is formed by high marked advantage because both positive levels of αAA, but these mutants still can and negative selections are possible. use αAA as a nitrogen source. Numerous Positive selection is carried out by lys2 mutants and low frequencies of lys5 auxotrophic complementation of the ura3 mutants can be conveniently obtained by and lys2 mutations, whereas negative simply plating high densities of normal selection is based on specific inhibitors, 5- cells on αAA medium. Similar with the fluoro-orotic acid (FOA) and α- FOA selection procedure, LYS2-containing aminoadipic acid (αAA), respectively, that plasmids can be conveniently expelled prevent growth of the prototrophic strains from lys2 hosts. Because of the large size but allows growth of the ura3 and lys2 of the LYS2 gene and the presence of mutants, respectively. numerous restriction sites, the FOA selection procedure with URA3 plasmids URA3 encodes orotidine-5’phosphate are more commonly used. decarboxylase, an enzyme which is required for the biosynthesis of uracil. 10.2 ADE1 and ADE2 Ura3- (or ura5-) cells can be selected on media containing FOA. The URA3+ cells The ADE1 and ADE2 yeast genes encode are killed because FOA appears to be phosphoribosylamino-imidazole- converted to the toxic compound 5- succinocarbozamide synthetase and fluorouracil by the action of phosphoribosylamino-imidazole- decarboxylase, whereas ura3- cells are carboxylase, respectively, two enzymes in resistant. The negative selection on FOA the biosynthetic pathway of adenine. Ade1 media is highly discriminating, and usually and ade2 mutants, but no other ade- less than 10-2 FOA-resistant colonies are mutants, produce a red pigment that is Ura+. The FOA selection procedure can be apparently derived from the used to produce ura3 markers in haploid polymerization of the intermediate strains by mutation, and, more importantly, phosphoribosylamino-imidazole (denoted for expelling URA3-containing plasmids, AIR). Furthermore, the formation of AIR including YCp and YEp replicating is prevented by blocks preceding the ADE1 plasmids, and integated YIp plasmids, as and ADE2 steps. For example ade2 strains discussed below for a number of genetic are red, whereas ade3 and the double strategies (Section 11). Because of this mutant ade2 ade3 are both white, similar negative selection and its small size, URA3 to the color of normal strains. Red colonies is the most widely used yeast marker in and red-white sectored colonies are easily yeast vectors. The specfic allele, ura3-52, detected by visual inspection.

21 The ade1 and ade2 red pigmentation, and inducer, including the action of repressors the prevention of the coloration by ade3 or at sites between the UAS and the TATA other ade- mutation has been incorporated box and the inhibition of galactose uptake. as a detection scheme in a number of Therefore, the addition of glucose to cells diversed genetic screens. Also, the ade2-1 growing in galactose meduim causes an UAA mutation, and the suppression of immediate repression of tramscription. The formation of the red pigment by the SUP4- UAS of galactose structural genes contains o suppressor has been used in a variety of one or more 17 base-pair palidromic genetic screens. Most of the screens are sequences to which Gal4p binds, with the based on the preferential loss, or the different levels of transcription determined required retention of a plasmid containing by the number and combinations of the a component involved in the formation of palidromes. the red pigment. The UAS of the divergently transcribed Examples of ade- red genetic screens GAL1 and GAL10 is contained within a include the detection of conditional 365-bp fragment, denoted PGAL1, that is mutations (Section 11.5, Plasmid Shuffle), sufficient for maximal galactose induction isolation of synthetic lethal mutations and thorough glucose repression. PGAL1 can (Section 12.5, Synthetic Enhancement and rapidly induce the expression of Epistatic Relationships), detection of YAC downstream fused-genes over 1000-fold transformants (Section 13.2, Yeast after the addition of galactose to cells Artificial Chromosomes [YACs]), and the growing in media with a nonfermentable isolation of mutations that effect plasmid carbon source. Furthermore, PGAL1 can be stability. turned off by the addition of glucose to the galactose containing medium. 10.3 GAL1 Promoter PGAL1 has been used in a wide range of Cloned genes can be expressed with studies with yeast, including the constitutive or regulatable promoters. The overproduction of yeast proteins as well as most commonly-used regulated promoter heterologous proteins (Section 13.3). Most for yeast studies is PGAL1. importantly, the strong glucose repression of PGAL1 has been used to investigate the There are two regulatory proteins, Gal4p terminal phenotype of essential genes, in and Gal80p, which effect the transcription much the same way that temperature shifts of the following structural genes: GAL1, a are used to control the activity of kinase; GAL2, a permease; GAL7, a temperature-sensitive mutations (see transferase; GAL10, an epimerase; and Section 11.2). Also, the PGAL1 system has MEL1, a galactosidase. Gal3p appears to been used to investigate suppression be required for the production of the (Section 12.4) and growth inhibition by intracellular inducer from galactose. In over expressed normal or mutant genes presence of the inducer, Gal4p binds to (dominant-negative mutations, Section sites in the UAS (upstream activation 12.1). PGAL1 is also an important sequence), and activates transcription. In component of one of the two-hybrid the absence of the inducer, such as when systems (Section 13.1). cells are grown in media containing nonfermentable carbon sources, Gal80p 10.4 lacZ and Other Reporters binds to the carboxyl terminal region of Gal4p, masking the activation domain. Activities of promoters, and protein- Expression is repressed in cells exposed to protein and protein-DNA interactions glucose-containing media for several involving promoter regions can be readily reasons in addition to the absence of the converted into selectable and quantifiable

22 traits by fusing the promoter regions to 11.1 Cloning by Complementation reporter genes. Reporter genes can be used to determine the levels of transcription, or Molecular cloning and DNA analysis is the the levels of translation of the transcript, most definitive way of characterizing a under various physiological conditions. gene that corresponds to a mutation. The most common use of reporter genes Cloning by complementation is usually has been to identify elements required for carried out with a library of a YCp vector transcription by systematically examining containing inserts of a more-or-less series of mutations in promoter regions. random series of genomic fragments, as Similarly, reporter genes have been used to illustrated in Figure 7.1 with the identify trans acting factors that modulate hypothetical yfg1 mutation. expression by transcription or translation. The yfg1 strain is transformed with the The Escherichia coli lacZ gene, which YCp library, and the transformants are encodes β-galactosidase, is the most examined for the Yfg+ trait. YCp vectors commonly used reporter with yeast and are generally used because each other systems, because its activity can transformant contains a single or only few assayed semiquantitatively on plates and plasmids per cell. The method of screening fully quantitatively by enzyme assay of transformants for complementation varies liquid cultures. Rare events can be detected according to the specific phenotype that is by the differental staining of colonies using to be scored. Direct selection can be used X-gal (5-bromo-4-chloro-3-indolyl-β-D- in some instances. However, if the original galactoside). mutation reverts, as is often the case, a high frequency of false positives occurs For positive selection, the reporter gene among the transformants. Thus, an could include, for example, the translated alternative method of indirect selection by region of the HIS3 gene, lacking a UAS replica-plating is preferred. Thus, by this (upstream-activating sequence). His+ method, the transformant containing the + colonies arise when active promoters are desired YCp-YFG1 plasmids appear as + formed, such as in the cloning of homogeneous Yfg colonies, whereas the heterologous components required for the colonies containing yfg1 revertants appear + - activation of a defined DNA segment. as heterogeneous Yfg and Yfg mixtures Combining the HIS3 selection with a lacZ after replica-plating. Most importantly, the screen is a commomly used strategy; this true transformants will be dependent on the + + approch of using two different reporters in YCp-YFG1 plasmid for the Yfg parallel with the same promoter region is phenotype. In almost all studies, plasmid an efficient means for identifying trans- dependency is conveniently determined acting factors. with the ura3 system and usually with the non-reverting allele ura3-52. Because ura3 11 Manipulating the Genome In mutants can be selected on FOA (5-fluoro- Vitro with Plasmids orotic acid) medium, plasmid-free strains therefore can be recovered and The greatest virtues of using yeast has been subsequently tested for the loss of complementation. For example, the yfg1 the ease with which genes can be retrieved, + + deleted, inserted and modified in a ura3 YCp-YFG1 strain would be Yfg Ura+, while the yfg1 ura3 strain, lacking controlled manner. These methods rely on - - the combined use of recombinant DNA the plasmid, would be Yfg Ura . techniques, transformation and classical Furthermore, the authenticity of the yeast genetics procedures. Overviews of plasmid can be confirmed by first some of these major approaches are recovering the plasmid in E. coli and described in the following sections. retransforming the yfg1 strain.

23 It is also desirable to confirm that the 11.2 Mutagenesis In Vitro cloned segment truly encompasses the YFG1+ gene. Even though the Two common experimental goals are to transformants may contain only a single produce either specific or "random" copy of the putative gene, there are mutations within a gene. DNA alteration examples in which two wild-type copies of are required for investigating, for example, a gene, one on the chromosome and the structure-function relationships and other on the plasmid, may suppress a essential regions of proteins, and for mutation situated at a different locus. An producing conditional mutations, such as independent test, based on homologous temperature-sensitive mutation. Specific recombination, relies on YIp vector alterations are carried out by the general containing the insert. If the insert contains procedure of oligonucleotide-directed a unique restriction site, cleavage at this mutagenesis that is applicable to any site will enhance integration of the plasmid cloned DNA segment, including those used at the homologous chromosomal site. for yeast studies. Oligonucleotide-directed Without cleavage, the plasmid could mutagenesis has been used to systemically integrate at the site of other yeast markers replace amino acids within proteins, on the plasmid, as well as at the YFG1+ especially the replacement of charged locus. After the integrant has been amino acids with alanine residues. Such obtained, the site of integration can be alanine replacements have resulted in a investigated by meiotic analysis. For multitude of effects, including proteins that example, integration of the p[YFG1+ were unaffected, inactive and temperature URA3+] plasmid at the site of YFG1+ locus sensitive. would result in a YFG1+::[YFG1+ URA3+] ura3 strain. After crossing to a yfg1 ura3 Also, numerous general procedures for strain and carrying out a meiotic analysis, producing "random" point mutations are the segregants should show a 2:2 available, including treating plasmid DNA segregation for both Yfg+/Yfg- and with hydroxylamine and misincorporation Ura+/Ura- and both markers would by PCR mutagenesis. Most importantly, a segregate as parental ditypes. On the other simple procedure has been developed for hand, if the plasmid integrated at a site the localized mutagenesis of yeast genes, other than the YFG1 locus, an excessive as illustrated in Figure 6B. The region to number of Yfg+ segregants would be be altered is first amplified under recovered, indicating that the normal mutagenic PCR conditions, resulting in the chromosomal YFG1+ allele and the generation of fragments containing integrated plasmid YFG1+ allele were not "random" yfg1-x mutations. A yfg1-∆ linked, or were at least not in close strain is then cotransformed with these proximity. PCR products and with a gapped YCp plasmid containing homology to both ends If the sequence of the YFG1 gene and of the PCR products. Repair of the gap flanking regions are known, the site of with the PCR products (see Section 11.6) integration could be confirmed solely by results in a series of strains with YCp PCR analysis. plasmids containing the altered yfg1-x alleles. The yeast strains containing the After the desired plasmid has been yfg1-∆ chromosomal deletion can then be demonstrated to encompass the YFG1 individually scored for the phenotype of gene, restriction fragments can be analyzed each of the yfg1-x mutants. This procedure to narrowed down the region of the locus, is particularly effective for targeting which can be subsequently sequenced and "random" mutations in specific regions, studied by a variety of other methods. and does not require subcloning steps in E. coli.

24 11.3 Two-step Gene Replacement The second cross-over can occur in either of two regions as depicted in Figure 11.1, After a gene has been cloned, the most the region either to the left or to the right efficient means for obtaining mutations in of the yfg1-1 alteration. The cross-over at the gene is by mutagenesis in vitro of the the left results in the regeneration of cloned DNA segment as described above. normal YFG1 allele, whereas a cross-over The effects of the mutations can then be at the right results in the introduction of examined in vivo by introducing the altered only the desired yfg1-1 mutation. gene in yeast by transformation. A simple and the most common procedure is to The position of the cross-over in the transform a yeast strain, which lacks a second step is approximately random, functional copy of the chromosomal gene, resulting in recovery of both YFG1 and with a YCp plasmid, which contains the yfg1-1 strains. However, the relative altered gene. This can be accomplished frequencies of cross-overs in the two directly in a single step if the gene in regions are probably related to their question is not essential. However, the best lengths. In order to recover the altered procedure, eliminating the problems of yfg1-1 allele, the second cross-over must copy number and vector sequences, is to occur at the opposite side of the site of replace the chromosomal copy of the gene integration. Therefore, it is desirable to with the altered plasmid copy. This can be force the initial integration at the smaller accomplished by the two-step gene region by cutting the plasmid in this region replacement procedure illustrated in Figure with a restriction endonuclease. 9.1. A YIp plasmid, containing the altered yfg1-1 gene is integrated in the In addition to the URA3 marker, the LYS2 chromosome in the region containing the can be also used for both positive and YFG1+ normal gene. Homologous negative selection (see Section 10.1, URA3 recombination results in two copies of the and LYS2). However, if neither URA3 or gene, yfg1-1 and YFG1+, separated by the LYS2 can be used, loop-out recombination plasmid sequences. The second step is often sufficiently high, 10-3 to 10-4, involves homologous crossing over in the making it possible to detect the loss of the repeated DNA segment to loop-out the marker by replica-plating. If a sufficiently plasmid, along with the URA3 gene. Such large number of altered replacements are desired Ura- strain can be selected on FOA contemplated, an additional marker could medium. The resulting plasmid is lost be introduced into the YFG1+ locus, during growth of the cells because the allowing for the convenient scoring of the plasmid lacks an origin of replication. desired loop-out.

25

Figure 11.1. (A) Retrieval of a chromosomal yfg1-1 mutation by transforming a mutant with a gapped plasmid. Only the repaired circular plasmid containing the mutation is stably maintained in yeast. (B) Generation of a series of yfg1-x mutations by PCR mutagenesis and gap repair. The yfg1-∆ mutant is cotransformed with the mutagenized PCR fragments and the gapped plasmid. The phenotypes of the yfg1-x mutants can be directly assessed in the strain containing the yfg1-∆ deletion.

11.4 Gene Disruption and One-step The one-step gene disruption procedure is Gene Replacement usually preferred because of its simplicity. This procedure is based on the use of a One of the most important and widely used linear fragment of DNA containing a methods to characterize yeast genes is gene selectable marker flanked by 5' and 3' disruption. The complete disruption of a homologous regions as illustrated in Figure gene unambiguously reveals its function 7A. The free ends of the fragment, and can be helpful for generating prepared by digestion with restriction additional mutations. Several methods can endonucleases, are recombinogenic, be used to produce deletions and null resulting in the integration of URA3 mutations, including the two-step gene marker and the loss of wild-type YFG1 replacement described above. allele.

26 It should be noted that the transformation A similar procedure has also been must be carried out in a diploid strain if the developed for conveniently replacing gene encodes an essential function. Also, mutant alleles in a single step, as illustrated the disruption of the desired genes should in Figure 11.2C. The YFG1 gene is first be verified by PCR or Southern blot disrupted with the URA3 gene as described analysis. The fragment required for single above. Replacements of the disrupted step disruptions can be also conveniently YFG1 by altered alleles can be selected on generated by PCR, alleviating the need to FOA medium among transformants or after clone the YFG1 gene. minimal growth of transformants on complete medium. This and related Because the one-step gene disruption procedures are particularly useful when procedure results in a URA3+ strain, the large numbers of replacements are method has been modified as illustrated in required. Figure 7B. In this method, URA3 is flanked by identical copies of the bacterial hisG Another method for producing gene gene (or any non-yeast DNA segment). disruptions, as well as simultaneously The hisG-URA3-hisG is first used to testing for the promoter activity, have been produce the gene disruption; subsequently, based on a dominant resistant module recombination between the direct repeats consisting almost entirely of heterologous and selection on FOA produces a single DNA. Transformants resistant to geneticin copy of hisG at the site of the disruption. (G418) are selected and examined for lacZ Thus, multiple rounds of gene disruption activity. To allow for repeated use of the can be carried out in the same strain. G418 selection, the module is flanked by short direct repeats, promoting excision in vivo.

27

Figure 11.2. Gene disruption and single-step gene replacement. (A) The YFG1+ gene is disrupted by transforming the strain with a linear fragment containing a URA3 selectable marker flanked by homologous sequences. The chromosomal segment is replaced by this URA3 containing fragment after integration by homologous recombination at the two ends. (B) The URA3 marker introduced in the YFG1 locus, can be excised if URA3 is also flanked by direct repeats of DNA, preferably not originating from yeast. Homologous recombinants, selection on FOA medium, lack the URA3 marker and retain a single copy of the repeated DNA. (C) Single-step gene replacement of mutant alleles, such as yfg1-1, can be carried out by first replacing the YFG1 gene by URA3, transforming the strain with linear fragment encompassing the yfg1-1 mutation, and selecting transformants on FOA medium, in which URA3 is replaced by yfg1-1.

11.5 Plasmid Shuffle to examine the phenotype of the transformants, yfg1-∆ p[yfg1-x]. However, As mentioned above (Section 11.2), the the characterization of mutations of an most common procedure for isolating and essential gene poses an additional technical characterizing a series of altered alleles, difficulty because of the inviability of yfg1-x, is simply to transform a strain strains containing the yfg1-∆ null mutation, lacking the gene, yfg1-∆, with YCp as well as of those containing plasmids containing the altered forms and nonfunctional yfg1-x alleles.

28

Figure 11.3. Plasmid shuffle. The chromosomal copy of YFG1 is replaced by the yfg1-∆ deletion, but the Yfg+ phen-otype is maintained by the YCp plasmid containing YFG1 and URA3. The strain is trans-formed with a mutagenized LEU2 plasmids having the YFG1 gene. Recessive yfg1-x mutations are manifested by selecting for strains on FOA medium. The strain will not grow on FOA medium if YFG1 is an essential gene and if the yfg1-x mutation is not functional.

Although the two-step gene replacement A haploid strain is first prepared, which procedure (Section 11.3) could be used to contains a YCp plasmid with the URA3+ generate condition yfg1-x mutations of an and YFG1+ gene and in which the essential gene, a method that more clearly chromosomal copy of the YFG1+ gene has reveals the nature of the yfg1-x mutation been deleted or disrupted (yfg1-∆). Such a has been developed, the so-called "plasmid strain could be prepared by transforming a shuffle" procedure as illustrated in Figure diploid strain that is hemizygous 11.3. (YFG1+/yfg1-∆) and choosing the

29 appropriate meiotic segregant with the also contains the ade3-∆ deletion, then the plasmid. adenine biosynthetic pathway is blocked before the step encoded by ADE2 and the The YFG1+ gene on YCp-LEU2 plasmid, strain is white (see Section 10.2, ADE1 and for example, is mutagenized and the ADE2). If ADE3 is carried by a YFG1+ resulting p[yfg1-x LEU2] plasmids plasmid, then the ade2-1 ade3-∆ strains are containing the yfg1-x alterations are red, but produce white sectors when the introduced into the yfg1-∆ p[YFG1 URA3] ADE3 plasmid is lost. The procedure does strain by transformation. Test on FOA not require replica-plating and is useful for medium then can be used to determine the detecting rare events. However, there are nature of the yfg1-x mutation. If the yfg1-x numerous other mutations that also allele was not altered or was completely produce white colonies, including ρ- functional, the p(YFG1 URA3) plasmid can mutations, resulting in relatively high be lost without preventing growth of the numbers of false positives. strain, which would appear to be FOA resistant (see Section 10.1, URA3 and 11.6 Recovering Mutant Alleles LYS2). On the other hand, if the yfg1-x allele was completely nonfunctional, the A convenient method for recovering strain will not grow on FOA medium. chromosomal mutations involves Furthermore, if the yfg1-x allele was transformation with gapped YCp plasmids, conditional, the growth on FOA medium as illustrated in Figure 11.1A. A double- would correspond to the condition. For stranded gap is produced by cleavage at example, a temperature-sensitive mutation two restriction sites within the cloned would be revealed by growth of the segment. The gapped plasmid is then used replica-plated colony on FOA medium at to transform a strain containing the desired 22°C but not 37°C; whereas the strain mutation that is encompassed in the would grow at both temperatures on chromosomal region corresponding to the complete medium lacking FOA. gap. The gapped plasmid is repaired with the homologous chromosomal region, The major disadvantage of the plasmid resulting in the capture of the yfg1-1 shuffle procedure, and other procedures mutation by the plasmid. The gapped using YCp plasmid, is that the copy plasmid is preferentially maintained, number varies from one to three or because only the circular form is possibly more copies per cell. Thus, replicated. The plasmid with the yfg1-1 different phenotypes may arise because of mutation can be subsequently recovered by the different levels of expression of the transforming E. coli with DNA from the altered gene product. Overexpression of yeast strain. As little as 100 base-pairs of some altered alleles can produce a nearly homology on either side of the gap is wild-type phenotype although a single sufficient to allow gap repair, although copy produces a mutant phenotype. A larger regions increase the efficiency of the more exact evaluation of a mutant allele process. may require the integration of a single copy with a YIp plasmid. 12 Interaction of Genes

Several variations of the plasmid shuffle Yeast genetics has been particularly procedure have been developed, and these amenable for identifying and rely on the production of a red pigment by characterizing gene products that directly certain adenine mutations. Strains having or indirectly interact with each other, mutations in the ADE2 gene (for example, especially when two mutations alleviate or ade2-1) accumulate a red pigment and enhance each otherïs defects. Information form red colonies. However, if the strain on a protein sometimes can be inferred

30 simply by examining the phenotypes of genetic terms used to denote the interaction haploid and diploid strains containing two of genes are summarized in Table 12.1, or more mutations. In addition, these using YFG1+, etc., as hypothetical genetic properties can be used for isolating examples. novel genes whose products interact. The

12.1 Heterozygosity and Dominant-negative Mutations

Table 12.1. Interactions of YFG (Your Favorite Gene) genes

Pheno- Genotype Ploidy Description type

YFG1+ 1n Yfg+ Wild-type dominant allele yfg1-1 1n Yfg- Nonfunctional, recessive mutation YFG1+ / yfg1-1 2n Yfg+ Heterozygous diploid yfg1-1 / yfg1-2 2n Yfg- Heteroallelic diploid yfg1-1 / yfg1- ∆ 1 2n Yfg- Hemizygous diploid

YFG1+ / yfg1-4 2n Yfg± Dominant-negative yfg1-4 mutation

+ - Dominant-negative overexpressed yfg1-4 YFG1 p[yfg1-4]N 1n Yfg mutation yfg1-1 / yfg1-3 2n Yfg± Intragenic complementation

YFG1+ yfg2-1 / yfg1-1 YFG2+ 2n Yfg+ Double heterozygous diploid YFG1+ yfg3-1 / yfg1-1 YFG3+ 2n Yfg± Non-complementation of a double heterozygous diploid suy1-1 1n Yfg+ Suppressor of yfg1-1 yfg1-1 suy1-1 1n Yfg+ Suppression of yfg1-1 by suy1-1 1n Yfg+ Suppression of yfg1-1 by overexpression of yfg1-1 p[YFG2+] N YFG2+ + + yfg1-1 PGAL1-YFG2 1n Yfg Suppression of yfg1-1 by overexpression of YFG2+ yfg1-4 1n Yfg± Partially functional mutation of YFG1+ yfg2-2 1n Yfg± Partially functional mutation of YFG2+ yfg1-4 yfg2-2 1n Yfg- Synthetic enhancement

31 When two recessive mutants are crossed in misfolding of proteins, dominant-negative a standard complementation test, the mutations retain at least portions of the phenotype of the resulting diploid strain structure, thus revealing specific critical usually reveals if the two mutations are regions. allelic and encode the same gene product. For example, if the yfg1-1 and yfg1-2 Dominant-negative mutations can also act mutations produce inactive Yfg1 proteins, in heterozygous diploid strains with one the diploid cross will be Yfg-. On the other copy of each allele. Such mutant proteins hand, if the two recessive mutations, yfg1- generally have a higher than normal 1 and yfg2-1, are in two different genes, affinity for a cellular component, and encoding two different polypeptide chains, displace the wild-type protein. For then the diploid cross, yfg1-1 YFG2+ x example, numerous nonfunctional CYC7 YFG1+ yfg2-1 would be Yfg+, because mutations were at least partially dominant both Yfg1p and Yfg2p are produced by the because the altered forms of cytochrome c wild-type alleles in the doubly were arrested at one of the steps in heterozygous diploid strain. mitochondrial import or heme attachment, and prevented entry of the normal form. As expected, mutations that inactivate a function are usually recessive. However, 12.2 Intragenic Complementation rare nonfunctional mutations can be dominant. Such dominant-negative One common exception in which mutations are particularly important heteroallelic diploid have a wild-type or because they can be used to identify near wild type phenotype is intragenic nonfunctional forms of the protein that complementation (also denoted allelic or retain their proper structure and associate intracistronic complementation) (Table with other cellular components. 12.1). If large numbers of pairwise crosses of independent mutations of a gene are As illustrated in Table 12.1, dominant- analyzed, complex complementation negative mutations can be revealed either patterns are often encountered, with some by overexpressing the mutation in a alleles showing complementation while haploid (or diploid) strain, such as YFG1+ others do not. For example, a yfg1-∆ p[yfg1-4]N, or by a single copy in deletion would not show intragenic heterozygous strains, such as YFG1+/yfg1- complementation with other yfg1-1 and 4. Most studies use multicopy YEp yfg1-3, although the yfg1-1/yfg1-3 cross plasmids for overexpressing mutations to could. Also, intragenic complementation uncover dominant-negative mutations. does not always restore the activity to the Similarly, the PGAL1 promoter fused to normal level and heteroallelic diploid mutant alleles, PGAL1-yfg1-4, could be used strains even can have conditional for the controlled overexpression in tests phenotypes. for dominant-negative mutations (see Section 10.3, GAL1 promoter). There are at least two mechanisms for intragenic complementation, one involving Dominant-negative mutations act by a proteins with two or more functional variety of mechanisms. For example, a domains, and the other involving proteins mutationally-altered transcriptional composed of two or more identical activator that retains DNA-binding polypeptide chains. activity, but lacks the ability to transactivate, could complex with the If a protein has two or more functional DNA-binding sites and displace the wild- domains that act independently, then a type protein. While most recessive missense mutation (an amino acid missense mutations produce an overall replacement) could inactivate one domain

32 without greatly effecting the others. Thus, though they are clearly functional in each partial or complete restoration could be of the singly heterozygous diploid strains observed in crosses of two such mutations, YFG1+/yfg1-1 and YFG3+/yfg3-1. Non- when each affected a different functional complementation of recessive non-allelic domain. As expected, intragenic mutations is only rarely encountered and complementation of this type is often may be the property of only certain observed with missense mutations, not proteins. with deletions, and only with special subsets of nonsense mutations. Numerous Several explanations can account for non- examples of intragenic complementation of allelic non-complementation. One can this type occurs with genes encoding consider two proteins Yfg1p and Yfg3p, amino acid biosynthetic enzymes, that carrying out related functions, and that including HIS4, which has three functional are at near limiting concentration in the domains. cell. In normal strains, both Yfg1p and Yfg2p are, by definition, at the normal Intragenic complementation is also 100% level, producing 100 units of each of observed with genes encoding proteins that the hypothetical proteins, with a total of are composed of two or more identical 200 units; in singly heterozygous strains, polypeptide chains. A haploid mutant the total would be 150 units, whereas, in could produce an abnormal polypeptide doubly heterozygous strains the total that assembles into an inactive would be 100 units. If the total level of 100 homomultimeric protein. On the other units for both proteins is below a critical hand, if two different abnormal threshold, than a mutant phenotype would polypeptide chains are produced in a be manifested in the doubly heterozygous heteroallelic diploid strain, the abnormal strains. Non-complementation of non- peptides could assemble in certain allelic gene has been observed for mutant combination to produce a catalytically gene encoding cytoskeletal proteins whose active multimeric protein. In such cases, cellular concentrations are critical for the abnormal polypeptide chains in some normal growth. way mutually compensates for each otherïs defect. Intragenic complementation of The lack of complementation of non-allelic genes encoding multimeric proteins is genes has also been explained by the surprisingly frequent among missense formation of inactive heteroligomers or mutations of certain genes. For example, 5 protein complexes, in which, for example, out of 10 genes controlling histidine both altered Yfg1p and Yfg3p biosynthesis show extensive independently assemble and inactivate the complementation, and the nature of the same protein complex, reducing its level genetic complementation maps suggest below a critical concentration. Also, that multimeric proteins may be involved. although recessive by themselves, these mutations may act like dominant-negative 12.3 Non-allelic Non-complementation mutation when in the doubly heterozygous condition, and encode abnormal proteins There are rare exceptions to the that actively compete or replace wild-type complementation of non-allelic genes, and subunits. these exceptions are denoted "non-allelic non-complementation" or "unlinked non- complementation". As illustrated in Table 12.1, certain recessive mutations of two different genes, yfg1-1 and yfg3-1 fail to complement in the doubly heterozygous diploid, YFG1+/yfg1-1 yfg3-1/YFG3+, even

33 12.4 Suppressors restricted subclass of mutants. For example, a temperature-sensitive mutation A suppressor is generally defined as a of actin gene was used to identify a mutation that completely or partially suppressor that was subsequently shown to restores the mutant phenotype of another encode an actin-binding protein. mutation. In the example given in the bottom of Table 12.1, the Yfg- phenotype In contrast, the suppressors that act of yfg1-1 mutation is restored by the indirectly on the same pathway are suppressor suy1-1. Suppressors can either expected to be gene specific, but allele have or not have a phenotype by non-specific, i.e., the suppressor could act themselves. on any allele, including null mutations, of a specific group of genes. Some of these are Suppressors an be broadly assigned to two denoted as "bypass" suppressor if they major groups, informational suppressors replace the function of the initial mutation. and metabolic suppressors. Informational For example, cyc1-∆ mutations, which suppressors encode either altered tRNAs or cause the deficiency of the major other components of the translational isocytochrome c, can be replaced by machinery, and act by misreading mRNAs. CYC7-H mutations, which act as For example, the nonsense suppressor, suppressors by overproducing the minor SUP4-o encodes an altered tyrosine tRNA isocytochrome c, and allows growth on that inefficiently inserts tyrosine residues lactate medium. Another mechanism for at UAA chain terminating codons because pathway suppression involves the loss of of an altered anticodon. Another class of regulation of successive steps, such as is informational suppressors are the so-called encountered, for example, in amino acid omnipotent suppressors which cause biosynthesis or the signal transduction ribosomal misreading because of pathways. If an upstream gene, that alterations in any one of a number of normally causes the activation of a proteins of the translational apparatus, downstream gene, is destroyed by including ribosomal proteins, elongation mutation, then mutant forms of the factors and release factors. Informational downstream gene can act as suppressors by suppressors characteristically act on certain rendering it independent of signalling from mutations of most, if not all genes, i.e., the upstream gene. they are allele specific but not gene specific. Another often-used means for uncovering interacting components of common On the other hand, metabolic suppressors functions is suppression by overexpression usually act on genes common to the same of wild-type alleles. As in the case for pathway or to a single metabolic function. suppression caused by mutation, While there are many possible mechanisms suppression caused by overexpression can for metabolic suppression, these occur by many different mechanisms. suppressors are now routinely isolated and Overexpression can be brought about with investigated as a means for identifying YEp multicopy plasmids, denoted as yfg1- + novel gene products of a pathway and for 1 p[YFG2 ]N in Table 12.1; or by fusion to identifying proteins that directly interact a strong inducible promoter such as PGAL1, + with each other. denoted as yfg1-1 PGAL1-YFG2 in Table 12.1. Furthermore, PGAL1 has the advantage The suppressors that act by direct physical that the activity can be conveniently turned interactions between two mutant proteins off by the addition of glucose to the are expected to be both gene and allele galactose containing medium, resulting in specific, i.e., the suppressors should act on the loss of suppression (see Section 10.3, only one or a few genes, and on only a GAL1 Promoter).

34 12.5 Synthetic Enhancement and double mutant would be inviable at any Epistatic Relationships temperature.

In some instances, the combination of two Synthetic enhancement is conceptually different mutant genes in a haploid strain identical to epistatic relationships. If two can enhance the severity of the phenotype mutant genes in a haploid condition more than when either of the mutant genes confers a phenotype that is quantitatively are by themselves. This exacerbation by identical as that conferred by each of the the combination of two genes, illustrated in single mutant genes alone, the two genes the bottom of Table 12.1 with the yfg1-4 are defined as being epistatic with respect and yfg2-2 alleles, have been denoted by a to one another. If, however, the doubly variety of terms in the early literature of mutant strain has an enhanced phenotype, genetics; currently, this phenomenon is the two genes are defined as being in two denoted "synthetic lethality" when cell separate epistasis groups. For example, growth is involved, or more generally, as detailed epistatic relationships of over "synthetic enhancement" or "synthetic thirty UV or ionizing radiation sensitive phenotypes". For example, two different mutants has revealed three non- mutant genes individually could cause overlapping epistasis groups. temperature-sensitive growth, but the

Figure 12.1. Synthetic enhancement and synthetic lethality. Novel chromosomal genes mutations, yfg2-1, etc., that enhance the phenotype of a yfg1-1 mutations can be uncovered by mutagenizing a yfg1-1 strain containing a YCp plasmid p[YFG1 URA3], and subsequently selecting for the loss of the plasmid. If yfg1-1 yfg2-1 strain are inviable, they will not grow on FOA medium.

As with suppression, synthetic There are numerous examples when enhancement can be caused by a number of synthetic lethality arise when two proteins mechanisms, but often the genes are are functionally redundant, such that associated with parallel or related neither nonfunctional form cause pathways controlling the same function, inviability, but both together are inviable. and some encode proteins that physically Synthetic enhancement of redundant genes interact with each other. is illustrated with strains having deletions of either of the CYC1 or CYC7 genes encoding, respectively, the two

35 isocytochromes c; single deletion strains 13 Genomic analysis are respiratory competent, while strains carrying both deletions are respiratory Many diverse studies require the deficient. Synthetic enhancement can be determination of the abundance of large indirect, as exemplified with mutations of numbers of specific DNA or RNA ARG4, encoding an arginine biosynthetic molecules in complex mixtures, including, enzyme and CAN1, encoding the arginine for example, the determination of the permease; the arg4 can1 double mutant changes in mRNA levels of many genes. does not grow on synthetic medium While a number of techniques have been containing arginine, while each of the arg4 used to estimate the relative abundance of and can1 single mutants do grow. two or more sets of mRNA, such as differential screening of cDNA libraries, As with suppressors, genes causing subtractive hybridization, and differential synthetic enhancement are commonly display, far more superior methods have isolated as means to identify genes in the been recently developed that are same or related pathways and ones particularly amenable to organisms whose encoding interacting proteins. Several entire genome sequences are known, such methods have been devised for this aim, as S. cerevisiae. It is now practicable to and one method is illustrated in Figure investigate changes of mRNA levels of all 12.1. A yfg1-1 ura3-52 strain, carrying a yeast ORFs in one experiment. YCp plasmid, p[YFG1+ URA3+], is mutagenized and tested on FOA medium. Numerous companies and academic groups A mutation, yfg2-1, causing synthetic have developed novel approaches to DNA enhancement is manifested in the Ura- sample preparation, probe synthesis, target strain because of the loss of the YFG1+ labeling and readout of arrays. The allele; in contrast, the other Ura- strains following procedures have been would show only the yfg1-1 phenotype. If successfully used for determining mRNA the yfg2-1 mutation causes synthetic levels in yeast: (i) the DNA Microarray lethality and the yfg1-1 yfg2-1 double System; (ii) the Oligonucleotide mutant is inviable, the colony would not Microarray System; (iii) the Low-density grow on FOA medium (see Section 10.1, DNA Array System; and (iv) the kRT-PCR URA3 and LYS2). System.

Other methods have been developed for The DNA Microarray System. As a general isolating mutations that confer synthetic means to address such problems as the lethality, similar to the detection schemes differential expression of an entire used with the plasmid shuffle procedures genome, Brown (1998) and his colleagues (Section 11.5). One of these other methods developed a system for making relies on the ade2 ade3 host mutations and microarrays of DNA samples on glass a YFG1 ADE3 plasmid, that results in red slides, probing the DNA micro-spots by sectoring when lost (see Section 10.2, hybridization with fluorescent-labeled ADE1 and ADE2). Another method is probes, and using a laser-scanning based on fusion of the GAL1 promoter, and microscope to detect the fluorescent screening PGAL1-YFG1 colonies for those signals corresponding to hybridization. that grow on galactose but not on glucose Fluorescent labeling allows for medium, presumably because of the simultaneous hybridization and separate presence of the desired yfg2-1 mutations detection of the hybridization signal from (see Section 10.3, GAL1 Promoter). two probes, thus allowing accurate determinations of the relative abundance of specific sequences in two complex samples. For example, with the DNA

36 Microarray System, the entire 6,400 ORFs fluorescently labeled cDNAs is detected by of the yeast S. cerevisiae can be placed on epifluorescence microscopy. This one slide. The mRNA levels of all ORFs technology was developed by the Affymax (open reading frames) can be determined Research Institute (Wodicka et al., 1997), after, for example, metabolic shifts or in and the oligonucleotide microarrays can be strains deleted for a single gene (DeRisi et produced only by the company. al., 1997). Using this technology, approximately The use of DNA Microarray System 6,200 or almost all ORFs of the entire S. requires the following basic steps for cerevisiae genome were probed for investigating gene expression of two differential mRNA expression in two related cell types or conditions: (i) growth conditions (16). Approximately preparation of the large set of DNA 12% of the ORFs, or approximately 750, elements, usually consisting of ORFs showed appreciable differential expression amplified by PCR with sets of primer pairs rates in the two growth conditions. specific for each ORF; (ii) preparation of DNA microarrays consisting of these The Low-density DNA Array System. In ORFs spotted on glass slides by a robotic addition to dotting on glass at high printing device, the Arrayer; (iii) densities, as described above for the DNA preparation of two related mRNAs derived Microarray System, sets of PCR-generated from cells that differ in the trait that is to ORFs can be spotted on nylon membranes, be investigated; (iv) preparation from the and hybridization to the dot blots can be mRNAs of fluorescently labeled cDNA by carried out with 32P-labeled cDNA for reverse transcription in the presence of detection of the relative levels of mRNAs. Cy3 (green) or Cy5 (red) labeled dUTP; (v) In some procedures, 1,536 dots were hybridization of the fluorescently labeled prepared on a single 9 x 13 cm positively cDNAs to the ORFs of the DNA charged nylon membrane. The lower microarrays printed on the glass slide; and density arrays on nylon membranes can be (vi) quantitative analysis of the relative achieved by automatic robotic and hand- abundance of the mRNAs from the degree held devices. No special equipment is of hybridization, using the Scanner. required to process the membranes once they are prepared. The DNA Microarray System requires two machines, the Arrayer and the Scanner. The kRT-PCR system. In addition to The Arrayer prints DNA samples screening for the differential expression of robotically onto a glass slide. After genes by the methods described above, hybridization, the Scanner analyzes the many investigations also require more two colored fluorescence of the array with accurate determinations of large number of a specially designed scanning confocal mRNA levels. While in the past, Northern microscope. blot analysis has been by far the most common means to quantitate mRNA The Oligonucleotide Microarray System. levels, new methods have been developed Modern photolithographic techniques are that are capable of producing more being used to generate miniaturized arrays accurate estimations of large number of of densely packed oligonucleotide probes. samples. Some of these are based on the These oligonucleotide microarrays, or automatic monitoring of reverse DNA chips, can then be used, for example, transcriptase initiated PCR (or kinetic for comparing two sets of cDNAs, similar monitored reverse transcriptase initiated to the DNA Microarray System described PCR, "kRT-PCR") . Several instruments above. Also, as with the DNA Microarray are commercial-available, such as the System, the hybridization pattern of LightCyclerTM and the ABI PrismTM 7700

37 Sequence Detection System, and some are into yeast cells has led to the development under development. The sensitivity, of methods for analyzing and preparing accuracy, and reproducibility of kRT-PCR DNA and proteins not only from yeast are remarkably high, allowing the itself, but also from other organisms. For detection of 20% differences and example, many mammalian homologs of quantitation raging from 367 to 0.00075 yeast genes have been cloned by using copies per cell of S. cerevisiae transcripts. heterologous cDNA expression libraries in yeast expression vectors. Also, yeast is being used to investigate the detailed functions of heterologous proteins, such as 14 Analyses with Yeast Systems mammalian transcription factors and nuclear hormone receptor. In fact, like E. The accessibility of the yeast genome for coli, yeast has become a standard genetic manipulation and the available microorganism for carrying out special techniques to introduce exogenous DNA tasks, some of which are described in this section.

Figure 14.1. The two-hybrid system. (A) Normally, the Gal4 transcription activator binds to DNA at the Gal4p binding sites and activates transcription of the lacZ reporter gene. (B) A hybrid of the Gal4 activation domain with the Yfg2 protein does not activate transcription because it does not localize at the Gal4 binding site. (C) A hybrid of the Gal4 DNA-binding site domain with the Yfg1 protein does not activate transcription of the reporter gene because of the lack of the transcriptional activation domain. (D) Protein-protein interaction between Yfg1p and Yfg2p reconstitutes Gal4p function and activates transcription of the reporter gene.

38 14.1 Two-hybrid Systems or partial genes are fused in frame with the GAL4 DNA-binding domain and the GAL4 Powerful methods, denoted two-hybrid transcription activation domains. If these systems, have been designed for screening two hybrid proteins interact, then the lacZ and investigating interacting proteins. reporter gene is transcribed, leading to the Because of the ease of the assay, blue color of the strain on medium contain exploratory two-hybrid screens are usually the chromogenic substrate X-gal (Figure the first method of choice when 10). In addition, yeast strains having not information of interacting proteins are only the PGAL1-lacZ but also the PGAL1- desired. HIS3 reporter genes are also available. It is advantageous to select directly for Some of these two-hybrid systems are expression of the PGAL1-HIS3 reported based on the properties of certain gene, followed by a screen for PGAL1-lacZ eukaryotic transcription factors, usually expression. Gal4p, that have two separate domains, one for DNA binding and the other for Another version of the two-hybrid system transcriptional activation. While the two uses the lexA operator sequence and the domains are normally on the same DNA-binding domain from the E. coli lexA polypeptide chain, the transcription factor repressor protein. In this system, the also functions if these two domains are activator domain is a segment of E. coli brought together by noncovalent protein- DNA that expresses an acidic peptide, protein interactions. In practice, gene which acts as a transcriptional activator in fusions are constructed so that the DNA- yeast when fused to a DNA-binding binding domain is linked to one protein, domain. As with the GAL4 system, lexA Yfg1p, and the activation domain is linked transcriptional activator also contains a to another protein, Yfg2p, as illustrated in nuclear localization signal that directs the Figure 14.1. Interactions of Yfg1p and protein into the nucleus. Yeast strains Yfg2p brings the DNA-binding and having lexA operators upstream of both the activation domains close together, leading E. coli lacZ and yeast LEU2 gene have to the expression of a reporter gene that is served as reporter genes. regulated by the transcription factor. Another two-hybrid system is based on the In addition, epitope tags have been built use of the lexA repressor protein and the into the constructs of both the GAL4 and lexA operator sequences from E. coli. lexA systems, allowing for the detection of These assays are almost always carried out expressed hybrid proteins. Although false- in yeast, although mammalian cells have positive and false-negative results can be been used. obtained, a substantial number of protein combinations have proved to be Yeast plasmid vectors are available, in successfully uncovered with the two- which the GAL4 DNA-binding domain and hybrid system and its use has become the GAL4 activation domain are on widely accepted. separate plasmids with convenient restrictions sites and with selectable yeast Because of its sensitivity, relatively low- markers. These plasmids are used in affinity interactions can be detected. Also, conjunction with reporter yeast strains, in the cloned genes encoding proteins that which upstream activation sequences from interact with the target protein becomes the GAL1-GAL10 region are used to immediately available when the two- promote transcription of the E. coli lacZ hybrid system is used in a screen with gene (the PGAL1-lacZ reporter gene) (see libraries of fused genes. Section 10.3, GAL1 Promoter, and Section 10.4, lacZ and Other Reporters). Complete

39 The two hybrid-system has been mainly YAC cloning systems are based on yeast used for the following three applications: linear plasmids, denoted YLp, containing testing proteins that are believed to interact homologous or heterologous DNA on the basis of other criteria; defining sequences that function as telomeres (TEL) domains or amino amino acids critical for in vivo, as well as containing yeast ARS interactions of proteins that are already (origins of replication) and CEN known to interact; and screening libraries (centromeres) segments. Manipulating for proteins that interact with a specfic YLp linear plasmids in vitro is complicated protein. The two hybrid-system has been by their inability to be propagated in E. successfully used to identify diversed sets coli. However, specially developed circular of interacting proteins in yeast and YAC vectors have been developed for mammalian cells, and it has been amplification in E. coli. For example, a particularly successful in studies of circular YCp vector, containing a head-to- oncogenes, tumor suppressors, protein head dimer of Tetrahymena or yeast kinases, and cell-cycle control. Some telomeres, is resolved in vivo after yeast examples of interacting proteins uncovered transformation into linear molecules with with the two hybrid-system in mammalian the free ends terminated by functional cells include Jun and Fos; Ras and the telomeres. One common type of YAC protein kinase Raf; the retinoblastoma vector that can be propagated in E. coli, protein or p53 and the SV40 large T contains telomeric sequences in inverted antigen; and other oncoproteins. orientation, which flank a DNA cassette containing the HIS3 gene (Figure 14.2). 14.2 Yeast Artificial Chromosomes After amplification in E. coli and before (YACs) transforming yeast the plasmid is digested with a restriction endonuclease, usually The initial step in the molecular BamHI, which excises the HIS3 cassette characterization of eukaryotic genomes and generates a linear form in vitro. Yeast generally requires cloning of large are transformed by this linear structure at chromosomal fragments, which is usually high frequencies, although the carried out by digestion with restriction transformants are unstable. Despite the endonucleases and ligation to specially presence of a CEN sequence, the YLp is developed cloning vectors. Usually 200 to present at high copy numbers and is lost at 800 kb fragments are cloned as Yeast high frequency because of its small size. Artificial Chromosomes (YACs), and 100- Increasing the size of the YLp by 200 kb fragments are cloned as Bacterial homologous integration in vivo or by Artificial Chromosomes (BACs or PACs). ligation in vitro increases the stability of The importance of YAC technology has the plasmid and reduces the copy number been heightened by the recently developed to approximately one per cell. methods for transferring YACs to cultured cells and to the germline of experimental animals.

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Figure 14.2. A yeast artificial chromosome (YAC) cloning system. The YAC vector contains telomeric ends that are denoted by black arrow heads. The vector also contains a unique SmaI cloning site flanked by SfiI and NotI 8-base-pair restriction sites. The vector can be used to clone 50 to 500 kb restriction fragments (see the text).

The developed highly-efficient YAC BamHI and SmaI, treated with alkaline cloning vectors also contain TRP1 and phosphatase and the two arms are ligated URA3 markers and a SUP4-o gene flanked to the exogenous DNA fragments desired by the NotI and SfiI rare restriction sites as to be cloned. A ade2-1 ura3-52 trp1-∆ host shown in Figure 14.2. The SUP4-o strain is transformed with the ligated suppressor also harbors a naturally mixture. Both arms are anticipated to be occurring SmaI site. The host strain present in Ura+ and Trp+ transformants and contains the ade2-1 UAA mutation, inserts should be present in the Ura+ Trp+ causing the formation of a red pigment, Ade- (red) transformants. YACs present in unless the mutation is suppressed by these transformants are then subjected to SUP4-o (see Section 10.2, ADE1 and pulse-field electrophoresis in order to ADE2). The YAC vector is cleaved with estimate the size of the inserts.

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Figure 14.3. Recombinational targeted cloning with YAC vectors. A yeast strain is transformed with a mixture of the two YAC vector arms and large fragments of DNA. Recombination in vivo results in the formation of a specific YAC clone. The two YAC vector arms are derived from linearized plasmids that contain targeting segments that are homologous to the termini of the DNA segment that is to be cloned.

The potential to use YAC cloning stem cells. Also, overlapping YAC clones technology has been enhanced by the can be recombined, resulting in larger ability to use homologous recombination clones encompassing more extensive for manipulating exogenous DNA in the regions. Furthermore, special YAC vectors yeast host. In recombinationally-targeted have designed for generating terminal and YAC cloning, YACs are assembled in internal deletions of cloned YAC inserts. vivo, by recombination, and not by ligation in vitro. Recombination takes place YACs have been useful for not only between a target segment of the exogenous cloning genes but also mammalian DNA, and the YAC vector that contain telomeric and centromeric regions, and sequences homologous to these targets as chromosomal origins of replication. illustrated in Figure 14.3. Transformation of the two YAC vectors arms and the 14.3 Expression of Heterologous exogenous segment, flanked by the target Proteins in Yeast segments, followed by recombination, results in the formation of the desired Although E. coli is still the first choice for stable YACs. The specific target DNA the producer of heterologous proteins, the segments for the YAC vector can be yeast S. cerevisiae has some attractive obtained from the exogenous DNA as features. Proteins produced in yeast, unlike restriction fragments or PCR products. those produced in E. coli, lack endotoxins. In certain special cases, such as hepatitis B Also YACs can be modified after cloning core antigen, the products produced in by "retrofitting", using homologous yeast have higher activity than those recombination with yeast plasmids having produced in E. coli. In contrast with using targeting sequences. For example, a E. coli, several posttranslational processing neomycin resistant gene has to be mechanisms available in yeast have incorporated into a YAC that will be allowed the expression of several human or transferred to mammalian cells using human pathogen-associated proteins with selection, as was done for transfering the appropriate authentic modifications. Such entire human β-globin gene in embryonic posttranslational modifications include

42 particle assembly, amino terminal ATG in the transcribed region of the acetylation, myristylation and proteolytic promoter, the heterologous gene must processing. In addition, heterologous provide an ATG that establishes the correct proteins secreted from specially reading frame corresponding to the amino- engineering strains are correctly cleaved terminus of the protein. It is essential that and folding and are easily harvested from this ATG corresponds to the first AUG of yeast culture media. The use of either the mRNA, because translation almost homologous or heterologous signal always initiates at the first AUG on peptides has allowed authentic maturation mRNAs from yeast as well as from other of secreted products by the endogenous eukaryotes. The 5’untranslated region of yeast apparatus. the vector also should be similar to the naturally-occurring leader region of The importance of yeast for production of abundant mRNAs by lacking secondary protein products by recombinant DNA structures and being A-rich, and G- methods is illustrated by the fact that the deficient, and by having an A at position -3 first approved human vaccine, hepatitis B relative to the ATG translational initiator core antigen, and the first food product, codon. rennin, were produced in yeast. Many of the expression vectors include a The cloning of specific cDNAs from other known signal for 3’ end formation of yeast organisms and the study of their function mRNA, although vectors lacking such using yeast as a surrogate does not defined signals synthesize transcripts until necessarily require high-level expression encountering a 3’-end forming signal from of the foreign protein. In these instances, another gene or a fortuitous signal on the the aim is just to produce physiological plasmid. quantities of the protein in a form that is correctly modified and localized in the cell Numerous normal and altered yeast such that the activity accurately reflects the promoters have been used, and these are activity in the original organism. However, chosen because of their high activity and commercial and laboratory preparations of some times because of their regulatory proteins generally require high expression properties. Some of the promoters have vectors. been derived from genes encoding alcohol dehydrogenase I, enolase, glyceraldehyde- There are numerous varieties of expression 3-phosphate dehydrogenase, vectors currently available for producing phosphoglycerate kinase, triose phosphate heterologous proteins in yeast, and these isomerase, galacokinase (PGAL1, see are derivatives of the YIp, YEp and YCp Section 10.3, GAL1 Promoter), repressible plasmids described above in Section 9. The acid phosphatase, α mating factor, etc. cDNA, synthetic DNA or genomic DNA These promoters almost always produce lacking introns are inserted in the vector. high levels of transcription of heterologous Promoters used in expression vectors gene, but there is a wide variation in the includes a transcription initiation site and amount of the corresponding proteins that variable amounts of DNA encoding the is finally produced in the yeast strain, 5’untranslated region. Because most of the depending on the specific heterologous yeast expression vectors do not contain an gene.

43 Key Words Plasmid shuffle, is a procedure for screening of mutations, Ascus, derived from a mutagenized plasmid, (plual asci) is a sac-like structure requiring the loss of a second plasmid to containing a tetrad of four spores (or assay for the recessive mutations. ascospores). Shuttle vectors, Heterothallic, are vectors that can be propagated in both strains of yeast have cross-compatible yeast and E. coli. mating types and are stable both as haploids and diploids. Tetrad, is the four products of meiosis. Homothallic, strains of yeast give rise to tetrads Two-hybrid system, containing four potentially self-fertile is a genetic assay used in yeast for members, because the transient haploid detection of protein-protein interactions. cells switch their mating types, and thus have only a stable diplophase. Bibliography

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