An Introduction to the Genetics and Molecular Biology of Yeast
An Introduction to the Genetics and Molecular Biology of Yeast
Table of Contents
1、Yeast is a Model Eukaryote (1)
2、Information on Yeast (2)
3、Yeast Strains (3)
4、Growth and Life Cycles (4)
5、The Yeast Genome (6)
6、Genetic Nomenclature (7)
6.1 Chromosomal Genes (7)
6.2 Mitochondrial Genes (9)
6.3 Non-Mendelian Determinants (11)
7、Genetic Analyses (11)
7.1 Overviews with Examples (11)
7.2 Tetrad analysis (13)
7.3 Non-Mendelian Inheritance (14)
8、Transformation (15)
8.1 Yeast Vector and DNA Fragments (15)
8.2 Synthetic Oligonucleotides (16)
8.3 Mitochondrial Transformation (16)
9、Yeast Vectors (17)
9.1 YIp Vectors (18)
9.2 YEp Vectors (19)
9.3 YCp Vectors (19)
10、Genes Important for Genetic Studies (20)
10.1URA3 and LYS2 (20)
10.2 ADE1 and ADE2 (21)
10.3 GAL1 Promoter (21)
10.4 lacZ and Other Reporters (22)
11、Manipulating the Genome In Vitro with Plasmids (22)
11.1 Cloning by Complementation (23)
11.2 Mutagenesis In Vitro (24)
11.3 Two-step Gene Replacement (24)
11.4 Gene Disruption and One-step Gene Replacement (26)
11.5 Plasmid Shuffle (27)
11.6 Recovering Mutant Alleles (29)
12、Interaction of Genes (29)
12.1 Heterozygosity and Dominant-negative Mutations (30)
12.2 Intragenic Complementation (31)
12.3 Non-allelic Non-complementation (32)
12.4 Suppressors (32)
12.5 Synthetic Enhancement and Epistatic Relationships (34)
13、Genomic analysis (35)
14、Analyses with Yeast Systems (37)
14.1 Two-hybrid Systems (37)
14.2 Yeast Artificial Chromosomes (YACs) (39)
14.3 Expression of Heterologous Proteins in Yeast (41)
Key Words (42)
Bibliography (43)
1、Yeast is a Model Eukaryote
This chapter deals only with the yeast S. cerevisiae, and related interbreeding species. The fission yeast Schizosaccharomyces pombe, which is only distantly related to S. cerevisiae, has equally important features, but is not as well characterized. The general principles of the numerous classical and modern approaches for investigating S. cerevisiae are described, and the explanation of terms and nomenclature used in current yeast studies are emphasized . This article should be particularly useful to the uninitiated who are exposed for the first time to experimental studies of yeast. Detailed protocols are described in the primary literature and in a number of reviews in the books listed in the Bibliography. The original citations for the material covered in this chapter also can be found in these comprehensive reviews.
Although yeasts have greater genetic complexity than bacteria, containing 3.5 times more DNA than Escherichia coli cells, they share many of the technical advantages that permitted rapid progress in the molecular genetics of prokaryotes and their viruses. Some of the properties that make yeast particularly suitable for biological studies include rapid growth, dispersed cells, the ease of replica plating and mutant isolation, a well-defined genetic system, and most important, a highly versatile DNA transformation system. Unlike many other microorganisms, S. cerevisiae is viable with numerous markers. Being nonpathogenic, yeast can be handled with little precautions. Large quantities of normal bakers’ yeast are commercially available and can provide a cheap source for biochemical studies.
Unlike most other microorganisms, strains of S. cerevisiae have both a stable haploid and diploid state. Thus, recessive mutations can be conveniently isolated and manifested in haploid strains, and complementation tests can be carried out in diploid strains. The development of DNA transformation has made yeast particularly accessible to gene cloning and genetic engineering techniques. Structural genes corresponding to virtually any genetic trait can be identified by complementation from plasmid libraries. Plasmids can be introduced into yeast cells either as replicating molecules or by integration into the genome. In contrast to most other organisms, integrative recombination of transforming DNA in yeast proceeds exclusively via homologous recombination. Exogenous DNA with at least partial homologous segments can therefore be directed at will to specific locations in the genome. Also, homologous recombination, coupled with yeasts’ high levels of gene conversion, has led to the development of techniques for the direct replacement of genetically engineered DNA sequences into their normal chromosome locations. Thus, normal wild-type genes, even those having no previously known mutations, can be conveniently replaced with altered and disrupted alleles. The phenotypes arising after disruption of yeast genes has contributed significantly toward understanding of the function of certain proteins in vivo. Many investigators have been shocked to find viable mutants with little of no detrimental phenotypes after disrupting genes that were previously assumed to be essential. Also unique to yeast, transformation can be carried out directly with synthetic oligonucleotides, permitting the convenient productions of numerous altered forms of proteins. These techniques have been extensively exploited in the analysis of gene regulation, structure-function
relationships of proteins, chromosome structure, and other general questions in cell biology. The overriding virtues of yeast are illustrated by the fact that mammalian genes are being introduced into yeast for systematic analyses of the functions of the corresponding gene products.
In addition, yeast has proved to be valuable for studies of other organisms, including the use of the two-hybrid screening system for the general detection of protein-protein interactions, the use of YACs for cloning large fragments of DNA, and expression systems for the laboratory and commercial preparation of heterologous proteins. Many of these techniques are described herein.
During the last two decades, an ever-increasing number of molecular biologists have taken up yeast as their primary research system, resulting in a virtually autocatalytic stimulus for continuing investigations of all aspects of molecular and cell biology. Most significantly, a knowledge of the DNA sequence of the complete genome, which was completed in 1996, has altered the way molecular and cell biologist approach and carry out their studies (see Dujon, 1996; Goffeau et al., 1996). In addition, plans are under way to systematically investigate the possible functions of all yeast genes by examining the phenotypes of strains having disrupted genes.
2、Information on Yeast
A general introduction to a few selected topics on yeast can be found in the book chapters "Yeast as the E. coli of Eucaryotic Cells" and "Recombinant DNA at Work" (Watson et al., 1987). Comprehensive and excellent reviews of the genetics and molecular biology of S. cerevisiae are contained in three volumes entitled "Molecular Biology of the Yeast Saccharomyces" (Broach et al., 1991; Jones et al., 1992; Pringle et al., 1997). An important source for methods used in genetics and molecular biology of yeast is contained in the book edited by Guthrie and Fink (1991). Overviews of numerous subjects are also covered in other sources (Broach et al., 1991; Brown & Tuite, 1998; Jones et al., 1992; Pringle et al.,1997; Wheals et al., 1995), including protocols applicable to yeasts (Fields & Johnson, 1993) and introductory material (Walker, 1998). A more comprehensive listing of earlier reviews can be found in Sherman (1991). Interesting and amusing accounts of developments in the field are covered in The Early Days of Yeast Genetics (Hall & Linder, 1992). The journal Yeast publishes original research articles, reviews, short communications, sequencing reports, and selective lists of current articles on all aspects of Saccharomyces and other yeast genera.
Current and frequently-updated information and databases on yeast can be conveniently retrieved on the Internet through World Wide Web, including the "Saccharomyces Genomic Information Resource"
(https://www.wendangku.net/doc/dd1729000.html,/Saccharomyces/) and linked files containing DNA sequences, lists of genes, home pages of yeast workers, and other useful information concerning yeast. From theMIPS page (http://www.mips.biochem.mpg.de/) you can access theannotated sequence information of the genome of Saccharomyces cerevisiae and view the chromosomesgraphically or as text, and more. The YPD page
(https://www.wendangku.net/doc/dd1729000.html,/YPDhome.html)contains a protein database with emphasis on the physical and functional properties of the yeast proteins.
3、Yeast Strains
Although genetic analyses and transformation can be performed with a number of taxonomically distinct varieties of yeast, extensive studies have been limited primarily to the many freely interbreeding species of the budding yeast Saccharomyces and to the fission yeast Schizosaccharomyces pombe. Although "Saccharomyces cerevisiae" is commonly used to designate many of the laboratory stocks of Saccharomyces used throughout the world, it should be pointed out that most of these strains originated from the interbred stocks of Winge, Lindegren, and others who employed fermentation markers not only from S. cerevisiae but also from S. bayanus, S. carlsbergensis, S. chevalieri, S. chodati, S. diastaticus, etc. Nevertheless, it is still recommended that the interbreeding laboratory stocks of Saccharomyces be denoted as S. cerevisiae, in order to conveniently distinguish them from the more distantly related species of Saccharomyces.
Care should be taken in choosing strains for genetic and biochemical studies. Unfortunately there are no truly wild-type Saccharomyces strains that are commonly employed in genetic studies. Also, most domesticated strains of brewers’ yeast and probably many strains of bakers’ yeast and true wild-type strains of S. cerevisiae are not genetically compatible with laboratory stocks. It is often not appreciated that many "normal" laboratory strains contain mutant characters. This condition arose because these laboratory strains were derived from pedigrees involving mutagenized strains, or strains that carry genetic markers. Many current genetic studies are carried out with one or another of the following strains or their derivatives, and these strains have different properties that can greatly influence experimental outcomes: S288C; W303; D273–10B; X2180; A364A; S1278B; AB972; SK1; and FL100. The haploid strain S288C (MAT a SUC2 mal mel gal2 CUP1 flo1 flo8-1 hap1) is often used as a normal standard because the sequence of its genome has been determined (Goffeau et al., 1996), because many isogenic mutant derivatives are available, and because it gives rise to well-dispersed cells. However, S288C contains a defective HAP1 gene, making it incompatible with studies of mitochondrial and related systems. Also, in contrast to S1278B, S288C does not form pseudohyae. While true wild-type and domesticated bakers’ yeast give rise to less than 2% r - colonies (see below), many laboratory strains produce high frequencies of r - mutants. Another strain, D273–10B, has been extensively used as a typical normal yeast, especially for mitochondrial studies. One should examine the specific characters of interest before initiating a study with any strain. Also, there can be a high degree of inviability of the meiotic progeny from crosses among these "normal" strains.
Many strains containing characterized auxotrophic, temperature-sensitive, and other markers can be obtained from the Yeast Genetics Stock Culture Center of the American Type Culture Collection
(https://www.wendangku.net/doc/dd1729000.html,/SearchCatalogs/YeastGeneticStock.cfm), including an almost complete set of deletion strains
(https://www.wendangku.net/doc/dd1729000.html,/cgi-bin/deletion/search3.pl.atcc). Currently this set consists of 20,382 strains representing deletants of nearly all nonessential ORFs in different genetic backgrounds. Deletion strains are also availabe from EUROSCARF
(http://www.uni-frankfurt.de/fb15/mikro/euroscarf/col_index.html) and Research Genetics (https://www.wendangku.net/doc/dd1729000.html,/products/YEASTD.php3). Other sources of yeast strains include the National Collection of Yeast Cultures
(https://www.wendangku.net/doc/dd1729000.html,/Sacchgen.html) and the Centraalbureau voor Schimmelcultures (http://www2.cbs.knaw.nl/yeast/webc.asp). Before using strains obtained from these sources or from any investigator, it is advisable to test the strains and verify their genotypes.
4、Growth and Life Cycles
Vegetative cell division of yeast characteristically occurs by budding, in which a daughter is initiated as an out growth from the mother cell, followed by nuclear division, cell-wall formation, and finally cell separation. The sizes of haploid and diploid cells vary with the phase of growth and from strain to strain. Typically, diploid cells are 5 x 6 μm ellipsoids and haploid cells are 4 μm diameter spheroids. The volumes and gross composition of yeast cells are listed in Table 1. During exponential growth, haploid cultures tend to have higher numbers of cells per cluster compared to diploid cultures. Also haploid cells have buds that appear adjacent to the previous one; whereas diploid cells have buds that appear at the opposite pole. Each mother cell usually forms no more than 20-30 buds, and it age can be determined by the number of bud scars left on the cell wall.
In addition, certain diploid strains of S. cerevisiae can assume a markedly different cell and colony morphology, denoted pseudohyphae, when grown on agar medium limiting for nitrogen sources. These pseudohyphal cells are significantly elongated, and mother-daughter pairs remain attached to each other. This characteristic pseudohyphal growth causes extended growth of branched chains outward from the center of the colony, and invasive growth under the surface of agar medium.
Table 4.1. Size and composition of yeast cells
cell Diploid cell
Characteristic Haploid
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 doubling time of approximately 90 min. in complete YPD (1% yeast extract, 2% peptone, and 2% glucose) medium and approximately 140 min. in synthetic media during the exponential phase of growth at the optimum temperature of 30°C. However, strains with greatly reduced growth rates
in synthetic media are often encountered. Usually strains reach a maximum density of 2 x108 cells/ml in YPD medium. Titers 10 times this value can be achieved with special conditions, such as pH control, continuous additions of balanced nutrients, filtered-sterilized media and extreme aeration that can be delivered in fermenters.
S. cerevisiae can be stably maintained as either heterothallic or homothallic strains, as illustrated in Figure 4.1. Both heterothallic and homothallic diploid strains sporulate under conditions of nutrient deficiency, and especially in special media, such as potassium acetate medium. During sporulation, the diploid cell undergoes meiosis yielding four progeny haploid cells, which become encapsulated as spores (or ascospores) within a sac-like structure called an ascus (plural asci). The percent sporulation varies with the particular strain, ranging from no or little sporulation to nearly 100%. Many laboratory strains sporulate to over 50%. The majority of asci contains four haploid ascospores, although varying proportions asci with three or less spores are also observed.
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 under control of a pair of MAT a/MATαheterozygous alleles, each ascus contains two MAT a and two MATα haploid cells. Upon exposure to nutrient condition, the spores germinate, vegetative growth commences and mating of the MAT a and MATα can occur. However, if the haploid spores are mechanically separated by micromanipulation, the haplophase of heterothallic strains can be stably maintained, thus allowing the preparation of haploid strains. In contrast, the presence of the HO allele in homothallic strains causes switching of the mating type in growing haploid cells, such that MAT a cells produce MATα buds and MATα cells produce MAT a buds. As a consequence, mating occurs and there is only a transient haplophase in homothallic strains (Figure 4.1).
Controlled crosses of MAT a and MATα haploid strains are simply carried out by mixing approximately equal amounts of each strain on a complete medium and
incubating the mixture at 30°C for at least 6 hr. Prototrophic diploid colonies can then be selected on appropriate synthetic media if the haploid strains contain complementing auxotrophic markers. If the diploid strain cannot be selected, zygotes can be separated from the mating mixture with a micromanipulator. Zygotes are identified by a characteristic thick zygotic neck, and are best isolated 4 to 6 hr after incubating the mixture when the mating process has just been completed.
5、The Yeast Genome
S. cerevisiae contains a haploid set of 16 well-characterized chromosomes, ranging in size from 200 to 2,200 kb. The total sequence of chromosomal DNA, constituting 12,052 kb, was released in April, 1996. A total of 6,183
open-reading-frames (ORF) of over 100 amino acids long were reported, and approximately 5,800 of them were predicated to correspond to actual protein-coding genes. A larger number of ORFs were predicted by considering shorter proteins. In contrast to the genomes of multicellular organsims, the yeast genome is highly compact, with genes representing 72% of the total sequence. The average size of yeast genes is 1.45 kb, or 483 codons, with a range from 40 to 4,910 codons. A total of
3.8% of the ORF contain introns. Approximately 30% of the genes already have been characterized experimentally. Of the remaining 70% with unknown function, approximately one half either contain a motif of a characterized class of proteins or correspond to genes encoding proteins that are structurally related to functionally characterized gene products from yeast or from other organisms.
Ribosomal RNA is coded by approximately 120 copies of a single tandem array on chromosome XII. The DNA sequence revealed that yeast contains 262 tRNA genes, of which 80 have introns. In addition, chromosomes contain movable DNA elements, retrotransposons, that vary in number and position in different strains of S. cerevisiae, with most laboratory strains having approximately 30.
Other nucleic acid entities, presented in Figure 5.1, also can be considered part of the yeast genome. Mitochondrial DNA encodes components of the mitochondrial translational machinery and approximately 15% of the mitochondrial proteins. ρo mutants completely lack mitochondrial DNA and are deficient in the respiratory polypeptides synthesized on mitochondrial ribosomes, i.e., cytochrome b and subunits of cytochrome oxidase and ATPase complexes. Even though ρo mutants are respiratory deficient, they are viable and still retain mitochondria, although morphologically abnormal.
The 2-μm circle plasmids, present in most strains of S. cerevisiae, apparently function solely for their own replication. Generally cir o strains, which lack 2-μm DNA, have no observable phenotype. However, a certain chromosomal mutation,
nib1, causes a reduction in growth of cir+ strains, due to an abnormally high copy number 2-μm DNA.
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 contain dsRNA viruses, that constitutes approximately 0.1% of total nucleic acid. RNA viruses include three families with dsRNA genomes, L-A, L-BC, and M. Two other families of dsRNA, T and W, replicate in yeast but so far have not been shown to be viral. M dsRNA encodes a toxin, and L-A encodes the major coat protein and components required for the viral replication and maintenance of M. The two dsRNA, M and L-A, are packaged separately with the common capsid protein encoded by L-A, resulting in virus-like particles that are transmitted cytoplasmically during vegetative growth and conjugation. L-B and L-C (collectively denoted L-BC), similar to L-A, have a
RNA-dependent RNA polymerase and are present in intracellular particles. KIL-o mutants, lacking M dsRNA and consequently the killer toxin, are readily induced by growth at elevated temperatures, and chemical and physical agents.
Yeast also contains a 20S circular single-stranded RNA (not shown in Figure 5.1) that appears to encode an RNA-dependent RNA polymerase, that acts as an independent replicon, and that is inherited as a non-Mendelian genetic element.
Only mutations of chromosomal genes exhibit Mendelian 2:2 segregation in tetrads after sporulation of heterozygous diploids; this property is dependent on the disjunction of chromosomal centromeres. In contrast, non-Mendelian inheritance is observed for the phenotypes associated with the absence or alteration of other nucleic acids described in Figure 5.1.
6、Genetic Nomenclature
6.1 Chromosomal Genes
The genetic nomenclature for chromosomal genes of the yeast S. cerevisiae is now more-or-less universally accepted, as illustrated in Table 6.1, using ARG2 as an example. Whenever possible, each gene, allele, or locus is designated by three italicized letters, e.g., ARG, which is usually a describer, followed by a number, e.g., ARG2. Unlike most other systems of genetic nomenclature, dominant alleles are denoted by using uppercase italics for all letters of the gene symbol, e.g., ARG2, whereas lowercase letters denote the recessive allele, e.g., the auxotrophic marker
arg2. Wild-type genes are designated with a superscript "plus" (sup6+ or ARG2+). Alleles are designated by a number separated from the locus number by a hyphen, e.g.,
arg2-9. The symbolΔ can denote complete or partial deletions, e.g., arg2-Δ1. Insertion of genes follow the bacterial nomenclature by using the symbol ::. For example, arg2::LEU2 denotes the insertion of the LEU2 gene at the ARG2 locus, in which LEU2 is dominant (and functional), and arg2 is recessive (and defective).
Table 6.1. Genetic nomenclature, using ARG2 as an example
Gene symbol Definition
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-Δ1 A specific complete or partial deletion of ARG2
ARG2::LEU2Insertion of the functional LEU2 gene at the ARG2 locus, and ARG2 remains functional and dominant
arg2::LEU2Insertion of the functional LEU2 gene at the ARG2 locus, and arg2 is or became nonfunctional
arg2-10::LEU2Insertion of the functional LEU2 gene at the ARG2 locus, and the specified arg2-10 allele which is nonfunctional
cyc1-arg2A fusion between the CYC1 and ARG2 genes, where both are nonfunctional
P CYC1-ARG2A fusion between the CYC1 promoter and ARG2, where the ARG2 gene is functional
Phenotypes are sometimes denoted by cognate symbols in roman type and by the superscripts + and -. For example, the independence and requirement for arginine can be denoted by Arg+ and Arg-, respectively. Proteins encoded by ARG2, for example, can be denoted Arg2p, or simply Arg2 protein. However, gene symbols are generally used as adjectives for other nouns, for example, ARG2 mRNA, ARG2 strains, etc.
Although most alleles can be unambiguously assigned as dominant or recessive by examining the phenotype of the heterozygous diploid crosses, dominant and recessive traits are defined only with pairs, and a single allele can be both dominant and recessive. For example, because the alleles CYC1+, cyc1-717 and cyc1-Δ1 produce, respectively, 100%, 5% and 0% of the gene product, the cyc1-717 allele can be considered recessive in the cyc1-717/CYC1+ cross and dominant in the
CYC1-717/cyc1-Δ1 cross. Thus, sometimes it is less confusing to denote all mutant alleles in lower case letters, especially when considering a series of mutations having a range of activities.
Although superscript letters should be avoided, it is sometimes expedient to distinguish genes conferring resistance and sensitivity by superscript R and S,
respectively. For example, the genes controlling resistance to canavanine sulphate (can1) and copper sulphate (CUP1) and their sensitive alleles could be denoted, respectively, as can R1, CUP R1, CAN S1, and cup S1.
Wild-type and mutant alleles of the mating-type locus and related loci do not follow the standard rules. The two wild-type alleles of the mating-type locus are designated MAT a and MATα. The wild-type homothallic alleles at the HMR and HML loci are denoted, HMR a, HMRα, HML a and HMLα. The mating phenotypes of MAT a and MATα cells are denoted simply a and α, respectively. The two letters HO denotes the gene encoding the endonuclease required for homothallic switching.
Dominant and recessive suppressors should be denoted, respectively, by three uppercase or three lowercase letters, followed by a locus designation, e.g., SUP4, SUF1, sup35, suf11, etc. In some instances UAA ochre suppressors and UAG amber suppressors are further designated, respectively, o and a following the locus. For example, SUP4-o refers to suppressors of the SUP4 locus that insert tyrosine residues at UAA sites; SUP4-a refers to suppressors of the same SUP4 locus that insert tyrosine residues at UAG sites. The corresponding wild-type locus that encodes the normal tyrosine tRNA and that lacks suppressor activity can be referred to as sup4+. Intragenic mutations that inactivate suppressors can denoted, for example, sup4- or sup4-o-1. Frameshift suppressors are denoted as suf (or SUF), whereas metabolic suppressors are denoted with a variety of specialized symbols, such as ssn (suppressor of snf1), srn (suppressor of rna1-1), and suh (suppressor of his2-1)
Capital letters are also used to designate certain DNA segments whose locations have been determined by a combination of recombinant DNA techniques and classical mapping procedures, e.g., RDN1, the segment encoding ribosomal RNA.
The general form YCRXXw is now used to designate genes uncovered by systematically sequencing the yeast genome, where Y designates yeast; C (or A, B, etc.) designates the chromosome III (or I, II, etc.); R (or L) designates the right (or left) arm of the chromosome; XX designates the relative position of the start of the
open-reading frame from the centromere; and w (or c) designates the Watson (or Crick) strand. For example, YCR5c denotes CIT2, a previously known but unmapped gene situated on the right arm of chromosome III, fifth open reading-frame from the centromere on the Crick strand.
E. coli genes inserted into yeast are usually denoted by the prokaryotic nomenclature, e. g., lacZ.
A list of gene symbols are tabulated in the book edited by Wheals et al. (1995), whereas a current list can be found in the Internet file
ftp://https://www.wendangku.net/doc/dd1729000.html,/pub/yeast/gene_registry/registry.genenames.tab
6.2 Mitochondrial Genes
Special consideration should be made of the nomenclature describing mutations of mitochondrial components and function that are determined by both nuclear and mitochondrial DNA genes. The growth on media containing nonfermentable substrates (Nfs) as the sole energy and carbon source (such as glycerol or ethanol) is the most convenient operational procedure for testing mitochondrial function. Lack of
growth on nonfermentable media (Nfs- mutants), as well as other mitochondrial alterations, can be due to either nuclear or mitochondrial mutations as outlined in Table 6.2. Nfs- nuclear mutations are generally denote by the symbol pet; however, more specific designations have been used instead of pet when the gene products were known, such as cox4, hem1, etc.
The complexity of nomenclatures for mitochondrial DNA genes, outlined in Table 6.2, is due in part to complexity of the system, polymorphic differences of mitochondrial DNA, complementation between exon and intron mutations, the presence of intron-encoded maturases, diversed phenotypes of mutations within the same gene, and the lack of agreement between various workers. Unfortunately, the nomenclature for most mitochondrial mutations do not follow the rules outline for nuclear mutations. Furthermore, confusion can occur between phenotypic designations, mutant isolation number, allelic designations, loci, and cistrons (complementation groups).
Table 6.2 Mitochondrial genes and mutations with examples
Wild-type
Mutation
(with examples)
Mutant phenotype or gene product
Nuclear genes
PET+pet- Nfs-
pet1 Unknown
function
cox4 Cytochrome
c oxidase subunit IV
hem1δ-Aminolevulinate synthase
cyc3 Cytochrome
c heme lyase Mitochondrial
DNA
Gross
aberrations
ρ+ρ-Nfs-
ρoρ- 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
tRNA Asp or M7-37 Mitochondrial tRNA Asp (CUG)
ant R Resistant
to
inhibitors
[ery S] ery R or [rib1] Resistant to erythromycin, 21S rRNA [cap S] cap R or [rib3] Resistant to chloramphenical, 21S rRNA [par S] par R or [par1] Resistant to paromomycin, 16S rRNA
[oli S] oli R or [oli1] Resistant to oligomycin, ATPase subunit
9
Nfs denotes lack of growth on nonfermentable substrates.
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 6.3. Table 6.3. Nomenclature of presumptive prions exhibiting non-Mendelian inhertance
Prion state Putative 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
7.1 Overviews with Examples
There are numerous approaches for the isolation and characterization of mutations in yeast. Generally, a haploid strain is treated with a mutagen, such as ethylmethanesulfonate, and the desired mutants are detected by any one of a number of procedures. For example, if Yfg- (Your Favorite Gene) represents an auxotrophic requirement, such as arginine, or temperature-sensitive mutants unable to grow at 37°C, the mutants could be scored by replica plating. Once identified, the Yfg-mutants could be analyzed by a variety of genetic and molecular methods. Three major methods, complementation, meiotic analysis and molecular cloning are illustrated in Figure 7.1.
Genetic complementation is carried out by crossing the Yfg- MATa mutant to each of the tester strains MATa yfg1, MATa yfg2, etc., as well as the normal control strain MATa. These yfg1, yfg2, etc., are previously defined mutations causing the same phenotype. The diploid crosses are isolated and the Yfg trait is scored. The
Yfg+ phenotype in the heterozygous control cross establishes that the Yfg- mutation is recessive. The Yfg- phenotype in MATa yfg1 cross, and the Yfg+ phenotype in the MATa yfg2, MATa yfg3, etc., crosses reveals that the original Yfg- mutant contains a yfg1 mutation.
Figure 7.1. General approaches for genetic analysis. As an example, a MAT a 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).
Meiotic analysis can be used to determine if a mutation is an alteration at a single genetic locus and to determine genetic linkage of the mutation both to its centromere and to other markers in the cross. As illustrated in Figure 7.1, the MATa yfg1 mutant is crossed to a normal MATa strain. The diploid is isolated and sporulated. Typically, sporulated cultures contain the desired asci with four spores, as well as unsporulated diploid cells and rare asci with less than four spores. The sporulated culture is treated with snail extract which contains an enzyme that dissolves the ascus sac, but leaves the four spores of each tetrad adhering to each other. A portion of the treated sporulated culture is gently transferred to the surface of a petri plate or an agar slab. The four spores of each cluster are separated with a microneedle controlled by a micromanipulator. After separation of the desired number of tetrads, the ascospores are allowed to germinate and form colonies on complete medium. The haploid segregants can then be scored for the Yfg+ and Yfg- phenotypes. Because the four spores from each tetrad are the product of a single meiotic event, a 2:2 segregation of the Yfg+:Yfg- phenotypes is indicative of a single gene. If other markers are present in the cross, genetic linkage of the yfg1 mutation to the other markers or to the centromere of its chromosome could be revealed from the segregation patterns.
The molecular characterization of the yfg1 mutation can be carried out by cloning the wild-type YFG1+ gene by complementation, as illustrated in Figure 7.1 and described below (Section 11.1 Cloning by Complementation).
7.2 Tetrad analysis
Meiotic analysis is the traditional method for genetically determining the order and distances between genes of organisms having well-defined genetics systems. Yeast is especially suited for meiotic mapping because the four spores in an ascus are the products of a single meiotic event, and the genetic analysis of these tetrads provides a sensitive means for determining linkage relationships of genes present in the heterozygous condition. It is also possible to map a gene relative to its centromere if known centromere-linked genes are present in the cross. Although the isolation of the four spores from an ascus is one of the more difficult techniques in yeast genetics, requiring a micromanipulator and practice, tetrad analysis is routinely carried out in most laboratories working primarily with yeast. Even though linkage relationships are no longer required for most studies, tetrad analysis is necessary for determining a mutation corresponds to an alteration at a single locus, for constructing strains with new arrays of markers, and for investigating the interaction of genes.
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).
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
:<1
Linkage >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 different chromosomes and are linked to their respective centromeres, there is a reduction of the proportion of T asci. If two genes are on different chromosomes and at least one gene is not centromere-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.
The frequencies of PD, NPD, and T tetrads can be used to determine the map distance in cM (centimorgans) between two genes if there are two or lesser exchanges within the interval:
The equation for deducing map distances, cM, is accurate for distances up to approximately 35 cM. For larger distances up to approximately 75 cM, the value can be corrected by the following empirically-derived equation:
Similarly, the distance between a marker and its centromere cM', can be approximated from the percentage of T tetrads with a tightly-linked centromere marker, such as trp1:
7.3 Non-Mendelian Inheritance
The inheritance of non-Mendelian elements can be revealed by tetrad analysis. For example, a cross of ρ+MAT a and ρ- MATα haploid strains would result in ρ+ MAT a/MATα and ρ-MAT a/MΑΤα diploid strains, the proportion of which would depend on the particular ρ- strain. Each ascus from a ρ+ diploid strain contains four ρ+ segregants or a ratio of 4:0 for ρ+:ρ-. In contrast, a cross of pet1MAT a and PET1+ MATα strains would result in a PET1+/pet1MATα/MAT a diploid, which would yield a 2:2 segregation of PET1+/pet1. Similar, the other non-Mendelian determinants also produce primarily 4:0 or 0:4 segregations after meiosis.
Another means for analyzing non-Mendelian elements is cytoduction, which is based on the segregation of haploid cells, either MAT a or MATα, from zygotes. Haploid cells arise from zygotes at frequencies of approximately 10-3 with normal
strains, and nearly 80% with kar1 crosses, such as, for example, kar1MAT a x KAR1+ MATα. While the haploid segregants from a kar1 cross generally retains all of the chromosomal markers from either the MAT a or MATα parental strain, the
non-Mendelian elements can be reassorted. For example, a MAT a canR1 kar1 [ρ-ψ-kil-o] x MATαCAN S1 [ρ+ψ+kil-k] cross can yield MAT a can R1kar1 haploid segregants that are [ρ+ ψ+kil-k], [ρ-ψ+kil-k], etc. In addition, high frequencies of 2 μm plasmids and low frequencies of chromosome can leak from one nucleus to another.
Also, the mating of two cells with different mitochondrial DNAs results in a heteroplasmic zygote containing both mitochondrial genomes. Mitotic growth of the zygote usually is accompanied by rapid segregation of homoplasmic cells containing either one of the parental mitochondrial DNAs or a recombinant product. The frequent recombination and rapid mitotic segregation of mitochondrial DNAs can be seen, for example, by mating two different mit- strains, and observing both Nfs-parental types as well as the Nfs+ recombinant (see Table 6.2).
8、Transformation
8.1 Yeast Vector and DNA Fragments
In general, transformation is the introduction into cells of exogenously added DNA and the subsequent inheritance and expression of that DNA. The most important advances in the molecular characterization and controlled modification of yeast genes have relied on the use of shuttle vectors which can be used to transform both yeast and E. coli.
The following three main methods are currently used to transform yeast: (i) those using spheroplasts; or (ii) cells treated with lithium salts; and (iii) the use of electroporation.
Spheroplasts for transformations are prepared by the action of hydrolytic enzymes to remove portions of the cell wall in the presence of osmotic stabilizers, typically 1 M sorbitol. Cell-wall digestion is carried out either with a snail-gut extract, usually denoted Glusulase, or with Zymolyase, an enzyme from Arthrobacter luteus. DNA is added to the spheroplasts, and the mixtures are co-precipitated with a solution of polyethylene glycol (PEG) and Ca2+. Subsequently, the cells are resuspended in a solution of sorbitol, mixed with molten agar and then layered on the surface of a selective plate containing sorbitol. Although this protocol is particularly tedious, and efficiency of transformation can vary by over four orders of magnitude with different strains, very high frequencies of transformation, over 104 transformants/mg DNA, can be obtained with certain strains.
Most investigators use cells treated with lithium salts for transformation. After treating the cells with lithium acetate, which apparently permeabilizes the cell wall, DNA is added and the cells are co-precipitated with PEG. The cells are exposed to a brief heat shock, washed free of PEG and lithium acetate, and subsequently spread on plates containing ordinary selective medium. Increased frequencies of transformation are obtained by using specially-prepared single-stranded carrier DNA and certain organic solvents.
A commonly-used method for transforming a wide range of different species of cells is based on the induced permeability to DNA by exposure to electrical fields. The interaction of an external electric field with the lipid dipoles of a pore configuration is believed to induce and stabilize the permeation sites, resulting in cross membrane transport. Freshly-grown yeast cultures are washed, suspended in an osmotic protectant, such as sorbitol, DNA is added, and the cell suspension is pulsed in an electroporation device. Subsequently, the cells are spread on the surface of plates containing selective media. The efficiency of transformation by electroporation can be increased over 100-fold by using PEG, single-stranded carrier DNA and cells that are in late log-phase of growth. Although electroporation procedures are simple, the specialized equipment and the required cuvettes are costly.
8.2 Synthetic Oligonucleotides
A convenient procedure has been described for producing specific alterations of chromosomal genes by transforming yeast directly with synthetic oligonucleotides. This procedure is easily carried out by transforming a defective mutant and selecting for at least partially functional revertants. Transformation of yeast directly with synthetic oligonucleotides is thus ideally suited for producing a large number of specific alterations that change a completely nonfunctional allele to at least a partially functional form. The oligonucleotide should contain a sequence that would correct the defect and produce the desired additional alterations at nearly sites. The method is apparently applicable to all mutant alleles whose functional forms can be selected. Although it is a general procedure, so far it has been extensively used only with mutations of CYC1, that encodes iso-1-cytochrome c, and CYT1 that encodes cytochrome c1. The transformation is carried out by the usual lithium acetate procedure, using approximately 50 mg of oligonucleotides that are approximately 40 nucleotides long.
8.3 Mitochondrial Transformation
Standard methods for transformation of nuclear genes are ineffective for mitochondrial DNA genes. However, DNA can be delivered to the mitochondrial matrix by high-velocity bombardment of yeast cells with tungsten microprojectiles carrying mitochondrial DNA. Several high-velocity microprojectile bombardment devices are commercially available, and these are powered by gunpowder charge or compressed gas.
This method was used to demonstrated that ro strains can be converted to stable "synthetic r-" strains by transformation with bacterial plasmids carrying mitochondrial genes (see Table 6.2). Similar to natural r- mitochondrial DNA, the synthetic r- mitochondrial DNA can recombine with r+ mitochondrial DNA, thus providing means to replace r+ wild-type genes with mutations generated in vitro.
Synthetic r- strains are isolated by bombarding a lawn of ro cells on the surface of a petri plate with YEp or YCp plasmids carrying both a selectable marker, such as URA3, and the mitochondrial gene of interest. The nuclear and mitochondrial genes may either be on separate or the same plasmid. Ura+ colonies, for example, are then
screen for the presence of the mitochondrial gene by crossing the colonies to an
appropriate mit- tester strain and scoring the diploids for Nfs+ (see Table 3). The
efficiency of mitochondrial transformation varies from experiment to experiment, and
can be from 2 x 10-3 to less than 10-4 mitochondrial transformants per nuclear
transformant.
9、Yeast Vectors
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
studies are shuttle vectors, which contain sequences permitting them to be selected
and propagated in E. coli, thus allowing for convenient amplification and subsequent
alteration in vitro. The most common yeast vectors originated from pBR322 and
contain an origin of replication (ori), promoting high copy-number maintenance in E.
coli, and the selectable antibiotic markers, the β-lactamase gene, bla (or Amp R), and
sometime to tetracycline-resistance gene, tet or (Tet R), conferring resistance to,
respectively, ampicillin and tetracycline.
In addition, all yeast vectors contain markers that allow selection of
transformants containing the desired plasmid. The most commonly used yeast
markers include URA3, HIS3, LEU2, TRP1 and LYS2, which complement specific
auxotrophic mutations in yeast, such as ura3-52, his3-Δ1, leu2-Δ1, trp1-Δ1 and
lys2-201. These complementable yeast mutations have been chosen because of their
low-reversion rate. Also, the URA3, HIS3, LEU2 and TRP1 yeast markers can
complement specific E. coli auxotrophic mutations.
The URA3 and LYS2 yeast genes have an additional advantage because both
positive and negative selections are possible, as discussed below (Section 10.1, URA3
and LYS2).
Table 9.1. Components of common yeast plasmid vectors
YCp Plasmid YIp YEp YRp
E. coli genes or segments
ori, bla; tet++++
Yeast genes or segments
URA3; HIS3; LEU2; TRP1; LYS2; etc. ++++
++0
leu2-d 0
2 μm; 2 μm-ori REP3; 0 +0 0
++
ARS1; ARS2; ARS3; etc. 0
+
CEN3; CEN4; CEN11; etc. 0
Host (yeast) markers
ura3-52; his3-Δ1; leu2-Δ1; trp1-Δ1; lys2-201; etc.++++
Stability +++±+
Although there are numerous kinds of yeast shuttle vectors, those used currently
can be broadly classified in either of following three types as summarized in Table 9.1:
integrative vectors, YIp; autonomously replicating high copy-number vectors, YEp;
or autonomously replicating low copy-number vectors, YCp. Another type of vector,