[ 1]
GETTING STARTED WITH YEAST
3
[1] Getting Started with Yeast
By FRED SHERMAN Virtues of Yeast The yeast Saccharomyces cerevisiae is recognized as an ideal eukaryotic microorganism for biological studies. Although yeasts have greater genetic complexity than bacteria, 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, a budding pattern resulting in dispersed cells, the ease of replica plating and mutant isolation, a well-defined genetic system, and, most importantly, a highly versatile DNA transformation system. Being nonpathogenic, yeast can be handled with few precautions. Large quantities of normal bakers' yeast are commercially available and can provide an inexpensive source for biochemical studies. Strains of Saccharomyces cerevisiae, unlike most other microorganisms, have both a stable haploid and diploid state, and they are viable with a great many markers. 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. Integrative recombination of transforming DNA in yeast proceeds exclusively via homologous recombinations, in contrast to most other organisms. Cloned yeast sequences, accompanied with foreign sequences on plasmids, can therefore be directed at will to specific locations in the genome. In addition, homologous recombination coupled with the high levels of gene conversion in yeasts has led to the development of techniques for the direct replacement of genetically engineered DNA sequences into 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 have contributed significantly toward understanding of the function of certain proteins in vivo. Many investigators have been shocked to find viable mutants with few or no detrimental phenotypes after disrupting "essential" genes. Genes can be directly replaced at high ettieieneies in yeasts and other fungi, but not in any other eukaryotie organisms. Also unique to yeast, transformation can be carried out directly with synthetic METHODS IN ENZYMOLO(3Y, VOL. 194
~ Š 1991 by A ~ l m i e Prm, 1 ~ All ~ l h ~ o f ~ o n i n ~ m y form~ed.
4
BASIC METHODS OF YEAST GENETICS
[ 1]
oligonucleotides, permitting the convenient production 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 routinely being introduced into yeast for systematic analyses of the functions of the" corresponding gene products. Furthermore, the replication of artificial circular and linear chromosomes has allowed detailed studies of telomeres, centromeres, length dependencies, and origins of replication. Mitochondrial DNA can now be altered in defined ways by transformation, adding to the already impressive array of genetic and biochemical techniques that have allowed detailed analysis of this organelle. The case with which the genome of yeast can be manipulated is truly unprecedented for any other eukaryote. Many of these techniques arc reviewed in this volume. Information on Yeast
A general introduction to a few selected topics on yeast can be found in other sources (see, e.g., Ref. 1). An article by Rine and Carlson2 outlines methods of yeast genetics with an emphasis on the use of recombinant DNA procedures. The extended survey of metabolism, cellular regulation, and cell division in S. cerevisiae by Hanes eta[. 3 contains 450 citations. Bennett et al. 4 recently reviewed the cell cycle, chromosome structure and function, and related topics concerning S. cerevisiae. Comprehensive reviews of the genetics and molecular biology of S. cer~isiae are contained in two volumes entitled "Molecular Biology of the Yeast Saccharomyces,"5 which are being revised and expanded.6 Overviews of numerous subjects J. D. Watson, N. H, Hopkins, J. W. Roberts, J. A. Stcitz, and A. M. Weiner, "Molecular Biology of the G-one," Chaps. 18 and 19. Benjamin/Cummings, Menlo Park, California, 1987. 2 j. Rine and M. Carlson, in "Gene Manipulation in Fungi" (J. W. Bennett and L. L. Lasure, eds.), p. 125. Academic Press, New York, 1985. 3 S. D. Hanes, R. Korcn, and K. A. Bostian, Crit. Rev. Biochem. 21, 153 (1986). 4 C. Bennett, E. Perkins, and M. A. Resnlck, in "Microbial Genetics" (U. Streipcs and R. Yasbin, cds.). Alan R. Liss, New York, 1990. s j. N. Strathern, E. W. Jones, and J. R. Broach (cds.), "Molecular Biology of the Yeast Saccharomyces," I: Life Cycle and Inheritance; II: Metabolism and Gcne Expression. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1981. 6 3. R. Broach, E. W. Jones, and J. R. Pringle (eds.), "The Molecular Biology of the Yeast Saccharomyces," Vols. 1 and 2. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1990.
[ 1]
GETTING STARTLEDWITH YEAST
5
are also covered in the volumes edited by Rose and Harrison, ~,' Prescott, 9 Spencer et al., '° Hicks, n and Barr e t a [ . , 12 as well as recent review articles. 13-n The text "Fungal Genetics" 19 covered general principles, especially regarding classical genetics of fungi, and contains a useful glossary. Many techniques useful in yeast studies have been covered in this series and elsewhere. 2°,2'
S t r a i n s o f Saccharomyces cerevisiae Although genetic analyses have been u n d e r t a k e n with a n u m b e r o f taxonomically distinct varieties o f yeast, extensive studies have been restricted primarily to the m a n y freely interbreeding species o f the budding yeast Saccharomyces a n d to the fission yeast Schizosaccharomyces pombe. Although the Latin b i n o m i a l Saccharomyces cerevisiae is c o m m o n l y used to designate m a n y o f the laboratory stocks o f Saccharomyces used t h r o u g h o u t the world, it should be pointed o u t that m o s t o f these strains originated f r o m the interbred stocks o f Winge, Lindegren, a n d others w h o e m p l o y e d fermentation m a r k e r s not only f r o m S. cerevisiae but also f r o m
S. bayanus, S. carlsbergensis, S. chevalieri, S. chodati, S. diastaticus, 7 A. H. Rose and J. S. Harrison (eds.), "The Yeasts," Volume I: Biologyof the Yeasts, 1969; Volume 2: Physiology and Biochemistry of Yeasts, 1971; Volume 3: Yeast Technology, 1970. Academic Press, New York. s A. H. Rose and J. S. Harrison (ecLs.),"The Yeasts," 2nd Ed., Volume 1: Biology of Yeasts, 1987; Volume 2: Yeast and the Environment, 1988. A_,~_d_q~aicPress, New York. 9 D. Prescott (ed.), "Methods in Cell Biology," VOl. 11, 1975; VOl. 12, 1976. Academic Press, New York. ,o j. F. T. Spencer, D. M. Spencer, and A. R. W. Smith (eds.), "Yeast Genetics, Fundamental and Appfied Aspects." Springer-Verla~ New York, 1983. " J . Hicks (ed.), "Yeast Cell Biology." Alan R. Liss, New York, 1986. 12p. j. Barr, A. J. Brake, and P. Valenzuela (eds.), "Yeast Genetics Engineering." Butterworth, Boston, 1989. t3 L. Guarente, Annu. Rev. Genet. 21, 425 (1987). ,4 M. Johnston, Microbiol. Rev. 51, 458 (1987). is I. Herskowitz, Microbiol. Rev. 52, 536 (1988). 16E. C. Friedberg,Microbioi. Rev. 52, 70(1988). ,7 F. Cross, L. H. Hartwell, C. Jackson, and J. B. Konopka, Annu. Rev. Cell. Biol. 4, 429 (1988). " IC Struhl, Annu. Rev. Biochem. .58, 1051 (1989). 19j. R. S. Fincham~ P. R. Day, and A. Radford, "Fungal Genetics." Univ. of California Press, Berkeley and Los Angeles, 1979. 20R. Wu, L. Grmsman, and IC Moldave (eds.), this series, Vol. 101. 2, I. Campbell and J. H. Duffus (eds.), "Yeast, a Practical A ~ h . " IRL Press, Oxford, 1988.
6
nASIC METHODS OF YEAST GENETICS
[ 1]
etc. 22~3 Nevertheless, it is still recommended that the interbreeding laboratory stocks of Saccharomyces be denoted as S. cerevisiae, in order to 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, probably many strains of bakers' yeast, and true wild-type strains of S. cer~isiae are not genetically compatible with laboratory stocks. It is usually not appreciated that many "normal" laboratory strains contain mutant characters, a fact not too surprising since they were derived from pedigrees involving mutagenized strains. The haploid strain $288C is often used as a normal standard because it gives rise to well-dispersed cells and because many isogenic mutant derivatives are available.23 However, $288C contains an abnormally low amount of cytochrome c and assimilates several carbon sources at reduced rates. In contrast to true wild-type and domesticated bakers' yeast, which give rise to less than 2% pcolonies (see below), many laboratory strains produce high frequencies of p- mutants. Another strain, D273-10B, has been extensively used as a typical normal yeast, especially for mitochonddal studies. One should examine the specific characters of interest before initiating a study with any strain. Many strains containing characterized auxotrophic, temperature-sensitive, and other markers can be obtained from the Yeast Genetics Stock Center [Donner Laboratory, University of California, Berkeley, CA 94720; (415)-642-0815]. Other sources of yeast strains include the American Type Culture Collection (12301 Parklawn Drive, Rockville, MD 20852), the National Collection of Yeast Cultures (Food Research Institute, Colney Lane, Norwich NR4 7UA, UK), the Centraalbureau voor Schimmelcultures (Yeast Division, Julianalaan 67a, 2628 BC Delft, Netherlands), and the Czechoslovak Collection of Yeasts (Institute of Chemistry, Slovak Academy of Sciences, Dubravaska cesta, 809 33 Bratislava, Czechoslovakia). Before using strains obtained from these sources or from any investigator, it is advisable to test the strains and verify their genotypes.
Genome of Saccharomycea cerewsiae
Saccharomyces cerevisiae contains a haploid set of 16 chromosomes that have been well characterized genetically 24and physically. 25-27A single 22C. C. Lindegren, "The Yeast Cell: Its Genetics and Cytology." Educational Publ., St. Louis, Missouri, 1949. 2J R. IC Mortimer and J. R. Johnson, Genetics 113, 35 (1986). 24R. IC Mortimer, D. Schild, C. R. Contopoloulou, and J. A. lCan~ Yeast 5, 321 (1989).
[ 1]
o E T r ~ o STARTED WITH YEAST
7
marker, KRB1, has been assigned to chromosome XVII,2s although there is no physical evidence for this chromosome. The 16 chromosomes range in size from 200 to 2200 kilobases (kb), with a total length of 14,000 kb. A total of 769 genes have been mapped, 24 constituting approximately onehalf of the reported mutations. The chromosomal genome is densely packed with an estimated 6500 genes having an average size of 2 kb and few introns. Ribosomal RNA is coded by approximately 120 copies of a single tandem array on chromosome XIL 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, listed in Table I,29-32 can also be considered part of the yeast genome. Mitochondfial DNA encodes components of the mitochondrial translational machinery and approximately 15% of the mitochondrial proteins, p° mutants completely lack mitochondrial DNA and are deficient in the respiratory polypeptides synthesized on mitochondrial ribosomes, namely, cytochrome b and subunits of cytochrome oxidase and ATPase complexes. Even though pO mutants are respiratory deficient, they are viable and still retain mitochondria, although morphologically abnormal ones. The 2-#m circle plasmids, present in most strains of S. cerevisiae, apparently function solely for their own replication. Generally cir ° strains, which lack 2-~tm DNA, have no observable phenotype; however, a certain chromosomal mutation, nibl, causes a reduction in division potential of cir + strains.33,34 Similarly, almost all S. cerevisiae strains contain double-stranded (ds) RNA viruses that are not usually extracellularly infective but are transmitted by mating. This dsRNA, constituting approximately 0.1% of total nucleic acid, determines a toxin and components required for the viral transcription and replication. K/L-o mutants, lacking class M dsRNA 2s S. L. Gerrin~ C. Conneny, and P. Hieter, this volume [4]. zs F. Sherman and L. P. Wakem, this volume [3]. 27p. P h i f i ~ A. Stotz, and C. Scherf, this volume [11]. 28 R. B. Wickner, F. Boutelet, and F. I-filger, MOl. Ceil. Biol. 3, 415 (1983). B. Dujon, in "Molecular Biology of the Yeast Saccharomyces IAfe Cycle and Inheritance" (J. N. Strathern, E. W, Jones, and J. R. Broach, eds.), p. 505. Cold Spring Harbor Laboratory, Cold ~ Harbor, New York, 1981. 3ej. R. Broach, this series, Vol. 101, p. 307. st G. F. Carle and M. Olson, Proc. Natl. Acad. Sci. U.S.A. 82, 3756 (1985). 3, R. B. Wickner, FASEB J. 3, 2257 (1989). 33C. Holm, Cell (Cambridge, Mass.) 29, 585 (1982). R. Sweeny and V. A. Zakian, Gowt/cs 122, 749 (1989).
8
BASIC METHODS OF YEAST GENETICS
[ 1]
(Table I) and consequently the killer toxin, are readily induced by chemical and physical agents, Only mutations of chromosomal genes exhibited Mendelian 2: 2 segregation in tetrads after sporulation Of heterozygous diploids19; this property is dependent on the disjunction of chromosomal eentromeres. In contrast, non-Mendelian inheritance is observed for the phenotypes associated with the absence or alteration of other nucleic acids described in Table I. Genetic Nomenclature Chromosomal Genes
The following genetic nomenclature for the yeast S. cerevisiae is now universally accepted. Gene symbols arc consistent with the proposals of Demeree et al., 3~ whenever possible, and are designated by three italicized letters (e.g., arg). Contrary to the proposals of Demerec et al. 35 for Escherichia coli, yeast genetic loci are identified by numbers, not letters, following the gene symbols (e.g., arg2). Dominant alleles are denoted by using uppercase italics for all letters of the gene symbol (e.g., ARG2). Lowercase letters denote the recessive allele (e.g., the auxotroph arg2). Wild-type genes are designated with the superscript plus symbol (sup6 + or ARG2+). Alleles are designated by a number separated from the locus number by a hyphen (e.g., arg2-14). Locus numbers should be consistent with the original assignments; however, allele numbers may be speeitie to a particular laboratory. For example, two different isolates from two different laboratories could be denoted can1-1, where both are mutations of the CAN1 locus. The symbol A can denote complete or partial deletions (e.g., his3-A1). Insertion of genes follows the bacterial nomenclature by using the symbol ::. For example, cycl::URA3 denotes the insertion of the URA3 gene at the CYC1 locus, in which URA3 is dominant (and functional) and cycl is recessive (and defective). 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. The following examples illustrate the conventions used in the genetic nomenclature for S. cerevisiae: ARG2 arg2 ARG2 +
Locus or dominant allele Locus or recessive allele that confers a requirement for arginine Wild-type allele
35M. Dernerce, E. A. Adelberg, A. J. Clark, and P. E. HartmCn; Genetics54, 61 (1966).
[ 1]
GETTING STARTED WITH YEAST
i
9
r~
u, o
I ee~
~6
O
Z
z
O
i ¢q
l0
BASIC METHODS OF YEAST GENETICS
arg2-9 Arg+ Arg-
[ 1]
Specific allele or mutation Strain not requiring arginine Strain requiring arglnine
Most alleles can be unambiguously assigned as dominant or recessive by examining the phenotype of the heterozygous diploid crosses. However, 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 +, cycl- 717, and cycl-A 1 produce, respectively, 100, 5, and 0% of the gene product, the cycl-717 allele can be considered recessive in the cycl-717/CYC1 + cross and dominant in the CYC1-717/cycl-A1 cross. Thus, sometimes it is less confusing to denote all mutant alleles in lowercase letters, especially when considering a series of mutations having a range of activities. There are a number of exceptions to these general rules. Gene clusters, complemention groups within a gene, or domains within a gene having a different characteristics can be designated by capital letters following the locus number;, for example, HIS4A, HIS4B, and HIS4C denote three regions of the HIS4 locus that correspond to three domains of the single polypeptide chain, which in turn determines three different enzymatic steps. Although superscript letters should be avoided, it is sometimes expedient to distinguish genes conferring resistance and sensitivity by the superscripts R and S, respectively. For example, the genes controlling resistance to canavanine sulfate (canl) and copper sulfate (CUP1) and their sensitive alleles could be denoted, respectively, as can~l, CUPRI, CANS/, and cupSl. 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 matingtype locus are designated MA Ta and MATa. The two complementation groups of the MATa locus are denoted MATal and MATo.2. Mutations of the MAT genes are denoted, for example, mata-I and matal-1. The wildtype homothallic alleles at the HAIR and HML loci are denoted HMRa, HMRot, HMLa, and HMLa. ~s Mutations at these loci are denoted, for example, hmra-1, hmla-1. The mating phenotypes of MA Ta and MATa cells are denoted simply a and a, respectively. 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, and sufl 1). 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 suppresI. Herskowitz and R. E. Jensen, this volume [8].
[ 1]
GETTING STARTED WITH YEAST
11
sors 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 coding for the normal tyrosine tRNA and lacking suppressor activity can be referred to as sup,l +. Intragenic mutations that inactivate suppressor can denoted, for example, sup4- or sup4.o-l. The nomenclature describing suppre~sor and wild-type alleles in yeast is unrelated to the bacterial nomenclature. For example, an ochre E. coli suppressor that inserts tyrosine residues at both UAA and UAG sites is denoted as su +, and the wild-type locus coding for the normal tyrosine tRNA and lacking suppressor activity can be referred to as Su4, su~, or supC. Frameshifl suppressors axe denoted as suf(or SUF), whereas metabolic suppressors are denoted with a variety of specialized symbols, such as ssn (suppressor of snfl), sm (suppressor of rnaI-I), and sub (suppressor of his2-1). Because the sites of mutations are usually used for genetic mapping, published chromosome maps usually contain the mutant allele. For example, chromosome III contains his4 and leu2, whereas chromosome IX contains SUP22 and FLDI. However, multiple dominant wild-type genes that control the same character (e.g.~SUCI and SUC2), axe used in genetic mapping, and such chromosomal loci are denoted in capital letters on genetic maps. Capital letters are also used to designate certain DNA segments whose locations have been determined by a combination of recombinant DNA techniques and classic mapping procedures, for example, RDN1, the segment encoding ribosomal RNA. Escherichia coli genes inserted into yeast are usually denoted by the prokaxyotic nomenclature (e.g., lacZ). In order to prevent duplications, new gene symbols should be approved by the committee for genetic nomenclature, headed by Dr. R. K. Mortimer [Department of Molecular and Cellular Biology, Division of Biophysics and Cell Physiology, University of California, Berkeley, CA 94720; (414)-643-8877].
Non-Mendelian Determinants Where necessary,non-Mendelian genotypes can be distinguishedfrom chromosomal genotypes by enclosure in square brackets (e.g.,[KIL-o] MATol trp1-1).Although it is advisable to employ the above rules for designating non-Mendelian genes and to avoid using Greek letters,the use of well-known and generally accepted Ga~ek symbolsshould be continued; thus, the original symbols p+, p-, q/+, and ~¢- or their transliteration, rho+, rho-, psi +, and psi-, respectively, should be retained. Detailed designations for mitochondrial mutants ~ and killer strains ss have been presented.
12
BASIC METHODS OF YEAST GENETICS
[1]
TABLE H SOME NoN-MENDELIAN DETERMINANTS OF YEAST a
Wild type
Mutant or polymorphic variant
p+
p-
K./L-k I
cir +
KIL-o cir 째
~/+
짜-
URE3
ure3-
Genetic element
Mutant phenotype
Mitochondrial DNA RNA plasmid 2-/zm circle plasmid Unknown Unknown
Deficiency of cytochromes a'a3, b, and respiration Sensitive to killer toxin None Decreased efficiency of certain suppression Deficiency in ureidosuccinate utilization
a Adapted from Refs. 29, 30, 32, 39, and 40. Other non-Mendelian determinants have been reported)4,4~
Some of the known non-Mendelian determinants in yeast are listed in T a b l e H. 29,3째,32,39,40
Growth of Yeast For experimental purposes, yeast are usually grown at 30 째 on the complete medium, YPD (Table III), or on synthetic media, minimal (SD) or complete (SC) (Tables IV 42 and V). For industrial or certain special purposes when large amounts of high titers are desirable, yeast can be grown in less expensive media with high aeration and pH control.43 The ingredients of standard laboratory media are presented in Tables III-VI. Synthetic media 42 are conveniently prepared with Bacto-yeast nitrogen base without amino acids (Difco Laboratories, Detroit, MI), containing the constituents presented in Table IV. Nutritional requirements of mutants are supplied with the nutrients listed in Table V. Growth on nonfermentable carbon sources can be tested on YPG medium (Table III), and 37L. A. Grivell, in "Genetic Maps" (S. J. O'Brien, ed.), p. 234. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1984. R. B. Wickner, in "Molecular Biology of the Yeast Saccharomyces Life Cycle and Inheritance" (J. N. Strathern, E. W. Jones, and J. R. Broach, eds.), p. 415. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1981. M. Aigle and F. Laeroute, Mol. Gen. Gem,t. 136, 327 (1975). 4oM. F. Tuite, P. M. Lund, A. B. Futcher, M. J. Dobson, B. S. Cox, and C. S. McLaughlin, PlasmidS, 103 (1982). 41 S. W. Liebman and J. A. All-Robyn, Curr. Genet. 8, 567 (1984). 42L. J. Wickersham, U.S. Dept. Agric. Tech. Bull., No. 1029 (1951). 43j. White, "Yeast Technology." Wiley, New York, 1954.
[I ]
OeTTrNO ST^RTED wrru X'E~ST
13
T A B L E HI
COMPLEXMEDIA Medium
Component
Comlx~ition
YPD (for routine growth)
1% Bacto-yeast extract 2% Bacto-peptone 2% Dextrose 2% Bacto-agar Distilled water
10 g 20 g 20 g 20 g 1000 ml
YPG [contain!he nonfermentable carbon source (glycerol) that does not support growth of por pet mutants]
1% Bacto-yeast extract 2% Bacto-peptone 3% (v/v) Glycerol 2% Bacta-agar Distilledwater
10 g 20 g 30 ml 20 g 970 ml
YPDG (used to determine proportion of p cells; p+ and p - colonies appear, ~ v e l y , large and small on this medium)
1% Bacto-yeast extract 2% Bacto-peptone 3% (v/v) Glycerol 0.1% Dextrose 2% Bacto-ag~ Distilledwater
10 g 20 g 30 ml 1g 20 g 970 ml
YPAD (for ~ t i o n of ~ u t s ; adenine is added to inhibit reversion of adel and ade2 mutatiomp
1% Bacto-yeast extract 2% Bacto-peptone 2% Dextrose 0.003% Adenine sulfate Distilled water 2% Bacto-agar
10 g 20 g 20 g 40 nag 10130 ml 20 g
Medium is dissolved in a boiling water bath, and 1.5-ml portions are dispensed with an automatic pipcttor into l-dram (3-ml) vials. The caps are screwed on loosely, and the vials are autoclaved. Atter autoclaving~ the rack is inclined so that the agar is just below the neck of the vial. The caps are tightened after 1 or 2 days.
fermentation markers can be determined with indicator media (Table VI) on which acid production induces color changes. Strains of S. cerevisiae can be sporulated at 30 ° on the media listed in Table VII; most strains will readily sporulate on the surface of sporulation medium after replica plating fresh cultures from a YPD plate, t* (Also see Refs. 45 and 46.) Media for petri plates are prepared in 2-liter flasks, with each flask containing no more than I liter of medium, which is sufficient for approximately 40 standard plates. Unless stated otherwise, all components are 44 R. R. Fowell, Nature (London) 170, 578 (1952). 4s B. Rockmi!!. E. J. Lambie, and G. S. Roeder, this volume [9]. 46 R. E. Fapmito, M. Dresser, and M. Breitenbach, this volume [7].
14
eASIC M~.rI~ODS OF YEASTGE~mTICS
[I]
TABLE IV S'rk-TtreTICMINIMAL(SD) MeDL*" Component
Composition
0.67% Bacto.yeast nitrogen base (without amino acids)
6.7 g
2% Dextrose 2% Bacto-agar Distilledwater
20 g
20 g I000 ml
Amount per liter Carbon source Dextrose Nitrogen source Ammonium sulfate Vitamins Biotin Calcium pantothenate Folic acid Inositol Niacin p-Aminobenzoic acid Pyridoxine hydrochloride Riboflavin Thiamin hydrochloride Compounds supplying trace dements Boric acid Copper sulfate Potassium iodide Ferric chloride Manganese sulfate Sodium molybdate Zinc sulfate Salts Potassium phosphate monobssic Potassium phosphate dibasic Magnesium sulfate
Sodium cldoride Calcium cldofide
20 g 5g 20/~g 2 mg 2/zg 10 mg 400/~g 200/~g 400/Jg 200/~g 400/~g 500/~g 40 #g 100/~g 200/Jg 400/J8 200/~8 400/J8 850 mg 150 mg 500 mg
100 mg 100 mg
o This synthetic medium is based on media described by Wickemham~ and is marketed, without dextrose, by Difco Laboratories (Detroit, MI) as "Yeast nitrogen base without Amilloacids." autoclaved together for 15 n a n at 250°F (i.e., 120°C) and 15 pounds (i.e., I atm) pressure. T h e plates should be allowed to dry at r o o m temperature for 2 - 3 days after pouring. The plates can be stored in sealed plastic bags
[ 1]
GETTING STARTED WITH YEAST
15
TABLE V S~rrHETIC COMrLE~ (SC) MeVtA~
Constituent Adenine sulfate Uracil L-Tryptophan L-Histidine-HC1 L-Arginine-HCl L-Methionine L-Tyrosine L-Leucine L-Isoleucine L-Lysine-HC1 L-Phenylalanine L--Glutami¢acid L-Aspartic acid L-Valine L-Threonine L-Serine
Final concentration (rag/liter)
Stock per 1000 ml
Amount of stock (mi) for 1 liter
20 20 20 20 20 20 30 30 30 30 50 100 100 150 200 400
200 m~' 200 mgb 1g 1g 1g 1g 200 mg 1g 1g 1g I gb 1 g~ 1 gb.C 3g 4 g0x 8g
10 10 2 2 2 2 15 3 3 3 5 10 10 5 5 5
° Synthetic complete medium contains synthetic minimal medium (SD) with various additions. It is convenient to prepare sterile stock solutions which can be stored for exte~ive periods. All stock solutions can be autoclaved for 15 min at 250°F. The appropriate volume of stock solutions is ~ to the ingredients of SD medium, and sufficient distilled water is added so that the total volume is I liter. The threonine and ~ acid solutions should be added separately after autoclaving. Concentrations of the stock solutions (amount per 100 ml) are given. Some stock solutions should be stored at room temperature in order to prevent pre~piration, whereas the other solutions may be refrigerated. It is best to use HCI salts of amino acids. b Store at room temperature. c Add after autoclavingthe media. for over 3 m o n t h s at r o o m temperature. The agar is omitted for liquid media. Different types o f synthetic media, especially omission media, can be prepared by mixing a n d grinding dry c o m p o n e n t s in a ball mill, Small batches o f liquid cultures can be grown in shake flasks using standard bacteriological techniques with high aeration. High aeration can be achieved by vigorously shaking cultures having liquid volumes less than 20% o f the flask volume. " N o r m a l " laboratory haploid strains have a doubling time o f 90 rain in Y P D m e d i u m a n d approximately 140 rain in synthetic media during the
16
BASIC METHODS OF YEAST GENETICS
[ 1]
TABLE VI INDICATORMEDIA
Indicator medium MALa
GALb
Component
Composition
1% Bacto-yeast extract 2% Bacto-peptone
10 g 20 g
2% Maltose Bromcresol purple solution (0.4% stock) 2% B a c t o - ~ r Distilled water
20 g
1% Yeast extract 2% Peptone
2% Agar Bromthymol blue(4 mg/mi stock) Distilled water 2% Galactose(20% stock)b
9 ml 20 g 1000 ml 10 g 20 g
20 g 20 mi 880 mi 100 ml
* Fermentation indicator medium used to distinguish strains which ferment or do not ferment maltose. Owing to the pH change, the maltose-fermenting strains will produce a yellow halo on a purple background. A 0.4% by dissolving 20 mg of the indicator bromcresol purple solution is ~ in 50 ml of ethanoL b Galactose-fermenting strains will produce a yellow halo on a blue background. After autoclaving, add 100 mi of a filter-sterilized 20% galactose
solution.
exponential phase of growth. However, strains with greatly reduced growth rates in synthetic media are often encountered. Usually strains reach a maximum density of 2 X 10s 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, filter-sterilization of media, and extreme aeration that can be delivered in fermentors. The sizes of haploid and diploid cells vary with the phase of growth4v and from strain to strain. Typically, diploid cells are 5 X 6 ~tm ellipsoids and haploid cells are 4/~m diameter spheroids. ~ The volumes and gross composition of yeast cells are listed in Table VIH. 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. 49 47 A. E. Wheals, in "~l'heYeast" (A. H. Rose and J. S. Harrison, eds.), 2nd Fed.,Vol. 1, p. 283.
Academic Press, New York, 1987. R. IC Mortimer, Radiat. Res. 9, 312 (1958). D. Freidfelder, J. Bacterio[. 80, 567 (1960).
[ 1]
GETTING STARTED WITH YEAST
[7
TABLE VII SPORULATIONMEDIA Sporulation medium
Component
Composition
Presl~orulationo
0.8% Baeto-yeast extract 0.3% Bacto-l~3tone 10% Dextrose 2% Bacto-agar Distilled water
0.8 g 0.3 g 10 g 2g 100 ml
Spornlation~
1% Potassium acetate 0.1% Bacto-yeast extract 0.05% Dextrose 2% Bacto-agar Distilled water
10 g 1g 0.5 g 20 g 1000 ml
Minimal sporulation~
1% Potassium acetate 2% Bacto-agar Distilled water
10 g 20 g 1000 ml
° Strains are grown 1 or 2 days on this medium before transferring to slmrulation medium. This is only necessary for strains that do not sporulate well when incubated on sporulation medium directly. b Strains will u n d e ~ o sevL~al divi~om on ~h]s medium and then sporulate after 3 to 5 days of incubation. Sporulation of auxotrophic diploids is usually incr~__._~lby adding the nutritional requirements to the sporutation medium at 25% of the levels given above for SD complete medium. Nutritional requirements are added as needed for auxotrophic diploids as for sporulation medium above (25% of level for SD complete medium).
TABLE VIII SIZE AND COMPOSITION OF YEAST CELLS
Characteristic Volume 0zm3) Composition (pg/ceilp Wet weight Dry weight DNA RNA Protein
Haploid cell 70 60 15 0.017 1.2 6
One picogram equals I0-l~ g.
Diploid cell ! 20 80 20 0.034 1.9 8
18
BASIC METHODS OF YEAST GENETICS
[ 1]
Strain Preservation Yeast strains can be stored for short periods of time at 4* on YPD medium in petri dishes or in closed vials (slants). Although most strains remain viable at 4" for at least 1 year, unusually sensitive mutants die after several months. Yeast strains can be stored indefinitely in 15% (v/v) glycerol at - 6 0 * or lower temperature. (Yeast tend to die after several years if stored at temperatures above -55".5°) Many workers use 2-ml vials (35 X 12 ram) containing 1 ml of sterile 15% (v/v) glycerol. The strains are first grown on the surfaces of YPD plates; the yeast is then scraped up with sterile applicator sticks and suspended in the glycerol solution. The caps are tightened and the vials shaken before freezing. The yeast can be revived by transferring a small portion of the frozen sample to a YPD plate. Other T e c h n i q u e s for Genetic Analysis Major techniques used for genetic analysis are covered in this volume, including dissection of asci, 51 gene mapping, 25~ and mutagenesis. 52-56 Other simple procedures used in yeast studies are described below. Replica Plating
Testing of strains on numerous media can be carried out by the standard procedure of replica plating with velveteen.57 However, subtle differences in growth are better revealed by transferring diluted suspensions of cells with specially constructed spotting apparatuses. An array of inoculating rods, fastened on a metal plate, is dipped into microtiter or other compartmentalized dishes, containing yeast suspensions. Small and uniform aliquots can be repetitively transferred to different types of media. Mating and Complementation
A few crosses can be simply carried out by mixing equal amounts ofthe MA Ta and MA Ta strains on a YPD plate and incubating at 30" for at least 6 hr and preferably overnight. Prototrophic diploid colonies can then be ~oA. M. Well and G. G. Stewart,Appl. Microbiol. 26, 577 (1973). ~ F. Shermanand J. B. Hicks,this volume[2]. ~2C. Lawrence,this volume[18]. ~3R. S. Sikorskiand J. D. Boeke,this volume[20]. M. F. Hoekstra, H. S. Seifert,J. Nickoloff,and F. Heffron,this volume[22]. "D. J. Garfinkeland J. N. Strathern,this volume[23]. R. P. Moerschell,G. Das, and F. Sherman,this volume[24]. ~7j. Lederbergand E. M. Lederberg,J. Bacteriol.63, 399 (I952).
[ 1]
GETTINGSTARTEDWITHYEAST
19
selected on appropriate synthetic media if the haploid strains contain complementing auxotrophic markers. Similarly, testing of mating types or other markers of meiotic progenies, which requires the selection of numerous diploid hybrids, can be carried out by replica plating, using any one of a number techniques such as cross-streaking and spotters. Prototrophic diploids also can be selected by overlaying a mixture of the two haploid strains directly on minimal plates, although the frequencies of matings may be slightly reduced. If the diploid strain cannot be selected, zygotes can be isolated from the mating mixture with a micromanipulator. Zygotes, which can be identified by a characteristic thick zygotic neck, are best isolated 4 - 6 hr after mixing~ when the mating process has just been completedss; diploids isolated by micromanipulation should be verified by sporulation and the lack of mating. Formation of prototrophic diploids, indicative of complementation, is used to test M A Ta and M A To~ mating types and to determine unknown markers in new mutants and meiotic segregants. Mating-type tests are best carried out with M A Ta and M A Ttx tester strains, each containing markers not in the strains to be tested. Complementation analysis consists of testing diploid strains that were constructed from two haploid mutants which have the same mutant phenotype, such as a specific amino acid requirement, sensitivity to UV light, or other mutant characters. If the mutant character is found in the diploid and if the two mutant genes are recessive, it can be concluded that the two mutant genes are allelic, that is, the lesions are in genes controlling the same function or, in most cases, the same polypeptide chain. In rare and special instances, a double heterozygous diploid strain may exhibit the phenotype of the recessive marker, confusing this test of complementation. However, because of allelic (or intragenic) complementation, the growth of double heterozygous diploids does not always indicate that the two mutations are in different genes. Some cases of allelic complementation occur when the normal enzyme is composed of two or more identical subunits. The enzymes formed by aUelic complementation are mutant proteins containing two different altered polypeptides in which each of the mutant polypeptides compensates for the defects of the other to produce a catalytically active protein. Allelic complementation can be pronounced when the enzyme contains separate domains carrying out different catalyric functions, such as the HIS4A, H I S 4 B , and H I S 4 C regions. Allelic complementation is frequent in yeast. For example, mutations in 5 out of 10 genes controlling histidine biosynthesis show extensive allelic complementation. Because of allelic complementation, frequencies of meiotic E. P. Sena, D. N. Radin, and S. Fogel, Proc. Natl. Acad. $ci. U.S.A. 70, 1373(1973).
20
BASIC METHODS OF YEAST GENETICS
[ 1]
recombination are required to determine if two complementing mutants are alleles of the same gene. The frequencies of recombination are extremely low if the mutations are in the same gene, whereas the frequencies of normal meiotic segregants can be as high as 25% if the mutations are in different genes. Complementation tests are required for scoring meiotic progeny from hybrids heterozygous for two or more markers controlling the same character. For example, a HIS3 + h i s 4 - Ă— his3- HIS4 + diploid will produce tetratype tetrads having the following genotypes: HIS3 + HIS4 + HIS3 + his4his3- HIS4 + his3- his4-
Intragenic complementation tests are required to determine the segregation of the his alleles in the HIS3 + his4-, his3- HIS4 +, and his3- his4segregants. These tests are carried out with MA Ta and MA Tot tester strains having either HIS3 + his4- or his3- HIS4 á markers. Diploids homozygous for either his3- or his4- will not grow on histidine-deficient medium as indicated in the tabulation below: XHIS3
+ his4-
Ă—his3-
HIS4 +
HIS3 + HIS4 +
+
+
+
HIS3 + his4-
-
-
+
his3- HIS4 +
-
+
-
his3- his4-
-
-
-
Random Spores
Although dissection of asci with recovery of all four aseospores is the preferred procedure for obtaining meiotic progeny,5m,52random spores can be used when isolating rare recombinants, or when analyzing a large number of crosses. Several techniques have been devised for eliminating or reducing unsporulated diploid cells from the culture. The proportion of random spores can be increased by sporulating the diploid strain on a medium containing a high concentration of potassium acetate (2%, w/v), which kills vegetative cells of many sporulated strains? 9 The sporulated culture is treated with Glusulase (snail juice) (NEN Research Products, Wilmington, Delaware), the spores are separated by a minimal level of sonication, and various dilutions are plated for single colonies. Furthers9 R. J. Rothstei n, R. E. Esposito, and M. S. Esposito, G e n e t i c s 85, 35 (1977).
[2]
MICROMANIPULATION
A N D DI$S]ECTION O F ASCI
~l
more, vegetative cells can be preferentially killed by treating a sporulated culture with an equal volume of diethyl ether.45 Another convenient method for producing random spores reties on the selection a~aln~t vegetative diploid cells that are heterozygous for cantl, the recessive marker confirming resistance to canavanine sulfate, e° If the canXl]+ diploid is sporulated and the spores are separated and plated on canavanine medium, one-half of the haploid spores will germinate and grow, whereas the remaining haploids and all of the diploids will not. Acknowledgments The writing of this chapter was supported by Public Health Research Grant GM12702 from the National Institutes of Health. ~0F. Sherman and H. Roman, Genetics 48, 255 (1963).
[2] M i c r o m a n i p u l a t i o n
a n d D i s s e c t i o n of Asci
By FRED SHERMANand JAMES HICKS Separation of the four ascospores from individual asci by micromanipulation is required for meiotic genetic analyses and for the construction of strains with specific markers. In addition, micromanipulation is used to separate zygotes from mass-mating mixtures and, less routinely, for positioning of vegetative cells and spores for mating purposes and for single-cell analyses. The relocation and transfer of ascospores, zygotes, and vegetative cells are almost exclusively carded out on agar surfaces with a fine glass microneedle mounted in the path of a microscope objective and controlled by a micromanipulator. Although specialized equipment and some experience are required to carry out these procedures, most workers can acquire proficiency with a few days of practice.
Micromanipulators Micromanipulators used for yeast studies operate with control levers or joysticks that can translate hand movements into synchronously reduced movements and microtools. ~ Most instruments were designed so that i F. Sherman, Methods Cell Biol. 11, 189 (1975). 2 H. M. EI-Bach'y, "Micromanipulators and Mieromanipulation." Academic Press, New York, 1963.
MIETHOI~ IN ENZYMOLOGY,VOL 194
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