[ 11 ]
[ 11 ] D N A o f S a c c h a r o m y c e s cerevisiae B y PETER PHILIPPSEN, AGATHE STOTZ,
Experimenters beginning to work with the DNA of Saccharomyces strains will find many of the current DNA isolation methods quite easy to follow. On the other hand, they may be confused by the pattern of bands seen following restriction endonuclease digestion and agarose gel electrophoresis of the DNA. The proper evaluation of these so-called restriction spectra is important for many experiments. Therefore, discussion of DNA isolation procedures is followed by a description of useful information derived from analysis of restriction spectra. The main focus is on DNA of Saccharomyces cerevisiae strains used in genetic studies. In addition, we describe our experience with domesticated strains or strains isolated from natural habitats and with species other than S. cerevisiae. D N A Isolation Procedures
The nucleus of a haploid S. cerevisiae cell contains approximately 14,000 kilobases (kb) of DNA subdivided into 16 linear chromosomal DNAs with sizes ranging from 250 to 2000 kb. !~ In addition, many strains carry 50 to 100 copies of a 6.3-kb plasmid which exists in two sequence arrangements and which is usually referred to as 2-/~m plasmid. 3 The 75 to 80 kb of mitochondrial DNA is circular, and each cell harbors 20 to 40 copies depending on the growth conditions.4 For most experiments preparations of total DNA can be used. Several published procedures for isolation of total DNA from Saccharomyces have been used successfully. We describe one procedure in detail that is adopted from several methods,S,~ and comment on the essential points. Volumes can be scaled up or down and, as with other rapid procedures, the DNA obtained is fragmented. However, the DNA is of sutficient length (50-250 kb) for most cloning and hybridization experiments. t G. F. Carle and M. V. Olson, Proc. Natl. Acad. Sci. U.S,4. 83, 3756 (1985). 2 S. L. Getting, C. Connelly, P. I-lieter, this volume . 3 j. R. Broach, in "The Molecular Biology of the Yeast Saccharomyces" (J. N. Strathern, E. W. Jones, and J. R. Broach, eds.), Vol. 1, p. 445. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1981. 4 T. D. Fox, L. S. Folley, J. J. Mulero, T. W. McMullin, P. E. Thorsness, L. O. Hedin, and M. C. Costanzo, this volume . 5 D. R. Cryer, R. Eccleshall, andJ. Marmur, Methods Cell. Biol. 12, 39 (1975). s F. Winston, F. Chumley, and G. R. link, this series, VoL 101, p. 211.
METHODS IN ENZYMOLOGY, VOL. 194
Copy0.gbt O 1991 by A~:~em~ Prm~, Inc. Allril~tsofrelmxlucl~n/n ~myfm-mre~rved.
CLONING A N D R E C O M B I N A N T
[I I ]
Basic Protocol 1. Grow cells in 30 ml YPD (2% yeast extract, 1% peptone, 2% dextrose) at 30 ° to a cell density of approximately 2 × l0 s cells/ml (early stationary phase). 2. Spin cells at 5000 rpm for 5 min in a Sorvall SS34 or similar rotor. Use 30-ml polypropylene tubes with screw caps. Centrifugation steps are performed at 4"; all other steps are carried out at room temperature unless otherwise specified. 3. Resuspend cells in 10 ml water and spin at 5000 rpm for 5 rain. 4. Resuspend cells in 3.0 ml of 0.9 M sorbitol, 0.1 M EDTA, 50 m M dithiothreitol, pH 7.5. Use the same tube until Step 9. 5. Add 0.5 mg DNase-free fl- 1,3-glucanase dissolved in 200 #1 0.9 M sorbitol (Zymolyase 20,000 from Seikagaku Kogyo, Ltd., Tokyo, Japan, or Miles Corp., Naperville, IL, or yeast lyric enzyme from ICN Biomedicals, Inc., Costa Mesa, CA) and incubate with occasional shaking at 37 ° . Conversion of the oval cells to round spheroplasts is followed with a fight microscope or is quantitatively monitored by mixing 50-gl aliquots taken at 10-min intervals with 950 gl of water and reading the ODs00 after 20 see. The conversion is complete when no more oval cells are visible or when the ODsoo has decreased by 80- 90%, respectively. This takes 15 - 120 min and depends on the strain used, on the growth medium, and on the growth phase. Alternatively, complete formation of spheroplasts can be checked by adding 100gl of 1% sodium dodecyl sulk'ate (SDS) to 100gl of the turbid suspension. A clear solution should form immediately. 6. Spin spheroplasts at 5000 rpm for 5 min in a Sorvall SS34 rotor and carefully discard the supernatant. 7. Resuspend spheroplasts in 3.0 ml of 50 m M Tris-HC1, 50 m M EDTA, oH 8.0, by slowly and repeatedly drawing the spheroplasts into a pipette and releasing them into the tube. Then mix with 0.3 ml of 10% SDS and incubate at 65" for 30 rain. 8. Add 1.0 ml of 5 M potassium acetate, mix, and let sit on ice for 60 min or longer. The white precipitate that forms consists mainly of insoluble potassium dodecyl sulfate and denatured proteins. 9. Spin at 15,000 rpm for 30 min and transfer the supernatant ( - 4 ml) to a 10- or 15-ml Corex tube or to a polypropylene tube. 10. Add 4.0 ml ice-cold absolute ethanol. On mixing, the nucleic acids (2% DNA and 98% RNA) and some residual proteins will immediately precipitate at room temperature. 11. Spin in Sorvall SM-24 or similar rotor at 10,000 rpm for 10 rain and discard the supernatant. Allow the salt to diffuse out of the pellet by incubation with 4.0 ml of 50% ethanol for at least 5 rain (vortex to break up the pellet). Spin at 10,000 rpm for 10 rain.
YEAS'r D N A
12. Discard the supernatant, dry the pellet under reduced pressure, and redissolve the DNA and RNA in 3.0 ml of 10 m M Tris, 1 m M EDTA, pH 7.5. This may take more than I hr. A 10-rain incubation at 42* can help. If a small pellet remains, spin at 10,000 rpm for 10 rain and transfer supernatant to a new tube. At this point a 10-#1 aliquot may be assayed by agarose gel electrophoresis as shown in Fig. 1B. 13. Add 150/zl of I mg/ml DNase-free pancreatic RNase (Boehringer Mannheim, FRG) and incubate at 37" for 30 rain. The stock of RNase is dissolved in 10 m M sodium acetate, pH 7.0, and kept a t - 20*. 14. Add 3.0 ml of 2-propanol, mix, and remove the precipitated DNA with a small plastic forceps, a glass hook, or by centrifugation. Wash the DNA with 50% 2-propanol and dry under reduced pressure. 15. Redissolve the DNA in 0.5 ml of 10 m M Tris-HCl, 1 m M EDTA, pH 7.5, and check the DNA by agarose gel electrophoresis as shown in Fig. IA. The yield should be 60-90/~g for a haploid strain. For quantitation, dilute 50 #1 with 0.95 ml of I0 m M Tris-HCl, 10 m M NaCI, pH 8.0, and measure the optical density at 260, 280, and 300 rim. If the ratio of OD~so/OD2m is 1.9 to 2.0 and the OD3o0 close to zero, an OD26o of 0.2 corresponds to 10/tg DNA per milliliter. The average fragment size of the DNA is 100 kb. The DNA should be stored at +4* or - 7 0 " , not at - 2 0 °. Comments Step I. The growth temperature for heat-sensitive strains is 20 ° to 23* and for cold-sensitive strains 30* to 35". When cells have to be grown in minimal medium or selective medium the cell density does not go beyond l0 s cells/ml in shaking flasks. Good yields are obtained when 50 m M sodium phosphate, pH 6.4, is added to the minimal medium. Cell densities can, in principle, be determined by measuring the O D e , of the culture. However, there is no universal correlation between cell density and O D e , since this correlation depends on the strain used and on the growth conditions. Step 4. The addition of thiol reagents such as dithiothreitol or 2-mercaptoethanol is not essential. They supposedly break S - S bridges in the cell wall and slightly decrease the time for spheroplast formation. Step 5. If cells start lysing (too much glucanase used) one may add one-tenth volume of 10% SDS, incubate 30 rain at 65 °, and proceed directly with Step 8. Insufficient conversion to spheroplasts will decrease the overall yield of DNA and sometimes leads to an overrepresentation of the 2-/~m plasmid. The glucanase is active between pH 6 and 9 with a pH optimum of 7.5. It can be inactivated by a 5-rain treatment at 60*. The glucanase preparation usually contains protease activity and traces of phosphatase activity but no DNase or RNase. The glucan cell wall of many
CLOmNO AtCD ReCOMSINAtCr D N A
[ 11 ]
Fla. 1. Separation of S. c~evisiae nucleic acids by dectrophoresis in 0.7% agarose gels. (A) aliquot of purified DNA (Step 15) run next to a mixture of ~ HindIII and ,1HindIlI-EcoRI fragments. Sizes, from top to bottom are as follows: 23.7 and 21.2 kb (one band), 9.42 kb, 6.56 kb, 5.15 and 4.97 kb (one band), 4.36 and 4.27 kb (one band), 3.53 kb, 2.97 kb (faint partial band), 2.32 kb, 2.03 kb, 1.90 kb, 1.58 kb, 1.37 kb, 0.95 kb, 0.83 kb, and 0.56 kb. This S. cerevisiae DNA prepaxatiott shows traces of fragments in the 10-25 kb range. In most preparations the nuclear DNA does not contain such small fr%ements, and the DNA band resembles that ofB. (B) DNA and RNA precipitated from the superuatant of Step 9 with half, equal, and double (left to fight) the volume of ethanol. After a short spin the pellets were dissolved and aliquots were run on the same gels as the DNA samples of A. The RNA is often less degraded at this step and, therefore, stains more intensely with ethidium bromide than shown here. Sometimes intact 18 S rRNA (1.75 kb) and 25 S rRNA (3.4 kb) are visible as strong bands.
yeasts including Ashbya, Candida, Hansenula, Kluyveromyces, Lipomyces, Pichia, Torulopsis, Saccharomycopsis, and Saccharomyces can be de-
graded. Step 7. It is essential to obtain a homogeneous and not too concentrated suspension of spheroplasts before the SDS is added.
YEAST D N A
Step 8. The slightly turbid solution contains in addition to the nucleic acids all soluble proteins, polysacchafides, lipids, and small molecular weight metabolites. Several procedures employ a treatment with I00/~g proteinase K at this step followed by repeated extractions with phenol, chloroform, and isoamyl alcohol (25: 24: 1). These extractions remove more material from the DNA solution than the precipitation with potassium acetate. However, the precipitation step is much more convenient, and the DNA obtained can be cleaved with all restriction endonucleases. Step 10. Despite common belief it is not necessary to add twice the volume of ethanol. The concentration of nucleic acids is so high that even half the volume of ethanol would lead to a complete precipitation of DNA and RNA as shown in Fig. 1B. Unwanted material which may precipitate with 66% ethanol will stay in solution. Step 11. Without removal of the salt it will take longer to dissolve the nucleic acid pellet and the double-stranded RNA may not be completely degraded by RNase (Step 13). Step 12. The separation on an ~ r o s e gel before treatment with RNase will show whether the strain carries the very abundant 4.2-kb linear double-stranded RNA or not. 7 The intensity of this RNA band may exceed that of the chromosomal DNA in ethidium bromide-stained gels. In addition the 2-~tm plasmid may be seen as a faint band ( Fig. 1B). Depending on the electrophoresis conditions, the supercoiled 2-~tm plasmid can comigrate with the 4.2-kb RNA. Step 13. The RNase treatment does not remove the RNA as often assumed. It converts the RNA to mono- and oligonucleotides by cleavage at pyrimidines. This allows a satisfactory separation of DNA from the vast excess of RNA by precipitation with 2-propanol. Small RNA molecules can be also separated from DNA by passage of the nucleic acid solution through BioGel AI5 (Bio-Rad, Richmond, CA). Step 15. In standard 0.7% ~ r o s e gel electrophoresis the uncleaved DNA should run as one band slightly slower than the 23.7-kb marker fragment of ~ DNA cleaved with HindIII (Fig. 1). Under these conditions DNA from 25 to over 500 kb migrates as one band. A faint fluorescence at the position of degraded RNA should not be underestimated with respect to RNA contamination since ethidium bromide intercalates only with the small fraction of RNA which is double stranded. Long-term storage at - 20 ° is not recommended because the salt in the DNA freezes out, and the freezing point of a saturated salt solution is about that of the cycling 7 R. B. Wickner, in "The Molecular Biology of the Yeast Saccharomyces" (J. N. Strathem, E. W. Jones, and J. R. Broach, eds.), VoL l, p. 415. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1981.
CLONING AND RECOMBINANT D N A
[ 11 ]
temperature o f - 20* freezers. Frequent freezing and thawing should be avoided.8 (Reference 8 also contains further valuable information for storing and processing DNA.) Alternative Small-Scale Isolation Procedure A very attractive modification for DNA isolations employs embedding of the washed cells in small blocks or beads of 1% low melting point agarose together with the glucanase.2,9-11 The spheroplasts are lysed in agarose by addition of SDS or sarcosyl followed by a treatment with proteinase K. The embedding in agarose allows separation ofthe immobile chromosomal DNA from other cellular material simply by washing the agarose particles. The DNA can be cleaved with restriction nucleases and also ligated to vector DNA in the solid or remolten agarose. Agarose without inhibitors for DNA-modifying enzymes is offered by several manufacturers (e.g., FMC BioProducts, Rockland, ME). Isolation and Fractionation of Yeast DNA by Cesium Chloride Gradients Equilibrium density gradients in CsCI yield exceptionally clean DNA. Treat cells from 300-ml cultures as described in the basic protocol up to Step 8. (Multiply volumes and ingredients by 10.) Extract twice with 10 ml phenol/chloroform/isoamyl alcohol (25:24: 1), adjust the aqueous phase to 0.5 M NaC1, and add the same volume of ethanol. Spin, wash the pellet in 50% ethanol, and dissolve the nucleic acids in 3 ml of 10 m M "Iris, 1 mM EDTA, pH 7.5. Do not treat with RNase since only large RNA separates well from DNA in CsC1 gradients. For isolation of ribosomal DNA (y-satellite) adjust the solution with CsC1 to a density of 1.70 g/ml (total volume 10-15 ml) and run in a fixed-angle rotor at 200,000 g for 60- 70 hr. The main nuclear DNA (40% G + C) and the 2-/tin plasmid band at 1.699 g/ml, the ribosomal DNA (56% G + C) at 1.704 g/ml, and the mitochondrial DNA (10% G + C) at 1.683 g/ml. s Although the rDNA cluster is part of chromosome XII, this DNA bands separately because of its slightly higher G + C content and because the chromosomal DNA is fragmented. For better separation of mitochondrial DNA from nuclear DNA, add bisbenzimide to the CsC1 gradients.4 For isolation of 2-#m plasmid DNA add ethidium bromide (final concentration 0.5 mg/ml) and adjust the solution with CsCI to 1.55 8 R. W. Davis, D. Botstein, and J. R. Roth, in "Advanced Bacterial Genetics," p. 211. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, 1980. 9 D. C. Schwartz and C. R. Cantor, Cell (Cambridge, Mass.) 37, 67 (1981). io D. A. Jackson and P. R. Cook, EMBO J. 4, 913 0985). t t M. N. K. Linskens and J. A. Huberman, Moi. Cell. Biol. 8, 4927 (1988).
YEAST D N A
g/ml. Run in swinging-bucket rotor at 150,000 g for 48-72 hr, isolate the plasmid (lower band) by side puncture with a syringe, and treat the DNA solution as in standard plasmid isolation procedures. It is always advisable to run the isolated DNA a second time in a CsCI gradient in order to achieve complete separation from RNA. Although RNA bands at higher density than DNA, traces of tRNA and small RNA fragments are still present in the DNA band after the first run.
Correlation between Bands in Restriction Spectra and R e p e a t e d D N A Sequences Approximately 85% of the sequences in yeast chromosomes are unique. Some genes exist in pairs, for example, genes for histones, c~-tubulin, translation elongation factor la, mating pheromones, several ribosomal proteins, and some giycolytic enzymes. Up to 15% of the nuclear DNA consists of repeated sequences, some of which can be seen as weak or strong bands after cleavage of the DNA with restriction endonucleases and separation of the fragments in agarose gels. These restriction spectra are specific not only for individual restriction endonucleases but also for the genomes of different Saccharomyces species. Interpretation of the band patterns can be quite useful. First, the extent of cleavage (partial or complete) can be deduced for many restriction endonucleases. Second, the average copy number of newly introduced plasmids can be estimated. Third, restriction site polymorphisms in repeated DNA can be detected. Figure 2 summarizes the sequence organization and the location of restriction sites for the four classes of repeated DNA (rDNA, 2-/~m plasmid, Ty elements, telomeric Y' sequences) which are the origin of most bands seen in restriction spectra. The ribosomal DNA consists of a cluster of usually 100 to 120 tandem copies o f a 9.08-kb repeat unit. ~2Cell clones with higher and lower copy number have been observed. ~3 The 2-#m plasmid is present in 50 to 100 copies in many but not all S. cerevisiae strains. We tested 50 domesticated and wild strains as well as laboratory strains and found 2-#m plasmids in 75% of the strains, occasionally with small deletions or loss of restriction sites. The two sequence arrangements shown for the 2-]~m plasmid originate from recombination events between the two inverted repeated sequences (IR). 3 Another class of repeated DNA are the mobile elements Tyl, Ty2, Ty3, and Ty4. ~4-16 Tyl and Ty2 are t2j. Warner,Microbioi. Rev. 53, 256 (1989). ~3G. F. Carle and M. V. Olson,personalcommunication. 14j. D. Boeke,in "Mobile DNA" (D. E. Bergand M. M. Howe, eds.), p. 335. American Societyfor Microbiology,Washington,D.C., 1989.
CLONINGAND RECOMBINANTDNA 9.08 kb
sm e .
EH P S ~
P~ BPs I/
B EE P
X Ps B Ps E
I1('1 IId 6.7 kb
,tl~S •~t., TI'~
SH KBaEB H
I i/ (~" 6,7 kb
Tyl (6.0 kb) 1 to 25 dispersed copies
Ty2 (6.0 kb) 1 to 12 dispersed copies
2 tt Plssmld (6.3 kb) 0 orS0 to 100 copies
8 H "[~" E
Xj~) EH P S ~
Ty3 (5.4 kb) 1 tO 4 dispersed copies ~ "~
Ty4(6.3kb) 1 tO 4 dispersed copies
If/f//'(GI"3T)nAR$ ~t TI
Y'repea~attelomeres Up tO 45 copies, single or In tandem
FiG. 2. Restriction maps of repeated nuclear DNA in S. cerevisiae. For the ribosomal DNA the arrangement of the rRNA coding sequences is shown. Arrows underneath the two repeat units represent the direction of transcription for the 5 S rRNA and the 35 S precursor rRNA. Arrows in the 2-/Jm plasmid indicate the relative orientation of the unique sequences (2.77 kb upper part and 2.36 kb lower part) between the 0.60-kb inverted repeats [J. L. Hartley and J. E. Donelson, Nature (London) 286, 860 (1980)]. The terminal direct repeats of Ty elements are shown as black bars, The black bars in the two telomeric Y' repeats mark the position of autonomously replicating sequences. The end of the chromosomes is determined by the repeated GI_3T sequence motif [J. J. Sahampay, J. W. Szostak, and E. H. Blackburn, Nature (London) 310, 154 (1984)]. Abbreviations for restriction sites are as follows: B for BgllI, Ba for BamHI, C for ClaI, E for EcoRI, H for HindlII, K for KpnI, P for PvulI, Ps for PstI, and X for XhoI.
closely related. Laboratory strains used in genetic studies usually carry 30 to 35 copies dispersed in the genome. However, we have found natural isolates carrying just one or a few copies. Ty3 and Ty4 are present only in 1 to 4 copies. ",~6 Ty elements can be lost from the chromosome by homolois D. J. Clark, V. W. Bilanchone, L. J. Haywood, S. L. Dildine, and S. B. Sandmeyer, J. Biol. Chem. 263, 1413 (1988). m6R. Stucka, H. Lochmfiller, and H. Feldmann, Nucleic Acids Res. 17, 4993 0989).
gous recombination between their 0.34-kb terminal direct repeats. Single copies of the terminal repeats remain at the previous location of the Ty element? 4 This explains the many copies of solo d sequences (50-100), solo a sequences (20-30), and solo • sequences (15-25) found in all strains even in those with low Ty copy number. The fourth class of repeated DNA visible in restriction spectra consists of the 6.7-kb telomeric Y' sequences? 7 These are present as single copies or short clusters of 2 to 4 copies at the ends of almost all 16 chromosomes in S. cerevisiae laboratory strains, with about 45 copies per haploid genome. Some natural isolates of S. cerevisiae carry very few copies of the Y' sequence, n Figure 3 shows examples of restriction spectra obtained with DNA of strain $288C, a strain commonly used in molecular and genetic studies. The DNA was isolated according to the procedure described above and was cleaved with five restriction endonucleases at two concentrations each. The EcoRI spectrum is dominated by ribosomal DNA fragments (R). 19Most S. cer~isiae stranm carry seven EcoRI sites in their rDNA. The site marked with an asterisk in the map of Fig. 2 is cleaved extremely slowly. Since the corresponding partial fragment of 0.94 kb (R*) is still visible in the spectrum, the cleavage was not quite complete. The second largest rDNA band (2.4 kb) varies slightly in size among S. cerevisiae strains owing to small deletions and insertions in the nontranscribed region adjacent to the 5 S rRNA gene.20 In some strains this fragment is fused to the 0.59 kb fragment because the EcoRI site 3' of the 25 S rRNA coding sequence is missing (see examples in Fig. 4). The four EcoRI fragments originating from the two forms of 2-/~m plasmids also are visible (p).2~ One of these comigrates with the 2.4-kb rDNA fragment. The two strong bands in the HindlII spectrum at 6.4 and 2.7 kb originate from rDNA. The DNA was cleaved to completion since no partial band at 9.1 kb is seen. Five bands with 2-/~m plasmid DNA also arc visible. 2t Each of the two plasmid isomeres carries three HindIII sites. Six fragments are generated, two of which arc identical, yielding the slightly more intense band at 1.3 kb. Y' sequences contain two HindIH sites 2.1 kb apart. The corresponding band runs close to the 2.2-kb plasmid band. The ~ C. S. M. Chan and B. K. Tye, Cell (Cambridge, Mass.) 33, 563 (1983). n D. Jt~gerand P. Philippsen, Mol. Cell. Biol. 9, 5754 (1989). 19Sizes of the rDNA EcoRI fragments are 2.79 (25 S rRNA), 2.46 (spacer and 5 S rRNA), 2.02 (18 S rRNA), 0.66 (18 S rRNA), 0.59 (25 S rRNA), 0.35 (25 S rRNA), and 0.22 kb (promoter region). 20 R. Jemtland, E. Maehlum, O. S. Gabrielsen, and T. B. Oyen, Nucleic Acids Res. 14, 5145 (1986). 2~The EcoRI fragments of the 2-/~m plasmid are 4.1, 3.9, 2.4, and 2.2 kb. The H/ndlII frasments of this plasmid are 4.1, 2.8, 2.2, 1.3, and 0.9 kb.
CLON~G AND RecoMs~A]~rr D N A
o o LU
BgllI spectrum shows one strong band at 4.55 kb which contains two rDNA fragments of equal size. The cleavage is complete since no partial band at 9.1 kb is seen. The 2-/zm plasmid is not cleaved with BgIII, and the open circles of plasmid monomers and plasmid dimers are visible as slowly migrating bands between the sample pocket and the largest linear DNA. Tyl elements carry three BglII sites, 1.65 and 2.25 kb apart. The corresponding fragments are seen as weak bands. Another weak band originates from the 1.7-kb BglII fragment in Y' sequences. The XhoI and BamHI spectra do not contain strong bands since neither~ enzyme cleaves in the rDNA or in the 2-#m plasmid. Uncleaved supercoiled and open circle forms of 2-~m plasmids are seen. The most prominent band (5.6 kb) in the XhoI spectrum originates from cleavage in the J sequences of Tyl and Ty2 elements. The intensity corresponds to 25 or 30 copies, taking into account that some of the approximately 35 Ty elements in this strain lack XhoI sites. The very weak band above the Ty band most likely represents the number of tandemly repeated Y' sequences. XhoI cleaves once in this sequence, as does BamHI. Indeed a band is present at the same position in the XhoI and the BamHI spectrum. No Ty-specific repeated fragments are expected to be seen with BamHI since this enzyme cleaves only a few Ty elements and in each case known only once. A few clearly visible bands were left unassigned. Some of these may originate from mitochondrial DNA. It is generally believed that this DNA becomes much more fragmented during the isolation procedure than nuclear DNA, and if bands carry mitochondrial DNA fragments their intensifies may therefore not reflect the number of mitochondrial genomes. Some unassigned bands could represent restriction site variants of known repeated sequences. There is ample of evidence for such variations in Ty elements and Y' sequences) 4:~ Other repeated DNA like tRNA genes (up to 12 dispersed copies for each of the 46 types oftRNAs) or the single J, a, and z sequences are too short to account for bands in the restriction spectra of Fig. 3. The restriction spectra of Saccharomyces species other than S. cerevisiae differ from those shown in Fig. 3. This is predominantly due to the
FIG. 3. Restriction spectra of DNA from S. cerevisiae strain $288C. About 1/~g of DNA was cleaved overnight with 1 and 5 units of the restriction nucleases indicated. The fragments were separated in a 0.7% agarosc gel and stained with 1/~g/ml ethidium bromide. The gel photo was cut into three parts to allow assignment o f b a n ~ originating from rDNA (R), 2-#m plasmids (P), Ty elements (Ty), or Y' sequences (Y'). The size marker lane between the EcoRI and HindlII spectra is the same as in Fig. 1. The DNA in the lelt lane of the BamHI spectrum appears uncleaved; probably much less than 1 unit BamHI was added. Further details are discussed in the text.
CLONIN~ AND RECOMBINANTDNA
expected sequence divergence among the genomes, which also affects restriction sites in repeated DNA. In addition, repeated sequences not essential for the life cycle (2-/~m plasmids, Ty elements, and Y' sequences) may be absent. The strongest bands in EcoRI restriction spectra represent almost exclusively rDNA, as concluded from hybridization experiments. Examples are shown in Fig. 4 for the proposed seven Saccharomyces species22 and a few other strains. In order to identify regions of high sequence conservation or high sequence divergence in the rDNA, the EcoRI spectra were hybridized with the total rDNA repeat unit of S. cerevisiae (Fig. 4A) and a 0.6-kb fragment carrying the 5 S rRNA gene and part of the nontranscribed spacer (Fig. 4B). Despite the variations all strains, even Kluyveromyces lactis, carry the two EcoRI sites close to the 3'-end of the 25 S rRNA coding sequence which generate the 0.35-kb fragment marked by the arrow. This extremely conserved region most likely plays an important role in ribosome assembly or function. The sequence of the spacer region which is represented by the second largest EcoRI fragment in S. cerevisiaerDNA (see Fig. 2) is not at all conserved, as evident from variations in length and intensity of those bands which hybridize to the spacer probe (Fig. 4B). Still, the weakly hybridizing bands are strong bands in the restriction spectra. Similar comparisons of singlecopy sequences in these Saccharomyces species often show no or only weak signals under stringent hybridization conditions, indicating that the average sequence divergence is 30% or more. This is in agreement with DNA renaturation studies performed before the tools of recombinant DNA techniques became available.23 When conserved genes are used as 22 j. A. Barnett, R. W. Payne, and D. Yarrow, "Yeast: Characteristics and Identification." Cambridge Univ. Press, Cambridge, 1983. 23j. N. Bicknell and H. C. Don~lag~J. Bacteriol. 101, 505 (1970).
FI6. 4. EcoRI fragments from ribosomal DNA of Saccharomyces and Kluyveromyces strains. About 1/zg total DNA was cleaved with EcoRl and separated on a 0.7% agarme gel. The fragments were denatured and blotted in both directions onto nitrocellulose. The left filter was hybridized with ~gt21-Sc310 carrying one S. cerevis/ae rDNA repeat [P. Philiplmen, M. Thomas, R. A. Kramer, and R. W. Davis, J. Mol. Biol. 123, 387 (1978)]. The right filter was hybridized with a subclone of ~t21-Sc310 which carries 0.6 kb of the 5 S rRNA gene and adjacent DNA. The size marker lane at left is the same as in F ~ 1; however, only fragments with homology to Agt21 are seen. The slowest mim'ath~band of S. telluris, S. douglasii, S. uvarum, and S. exiguus rDNA represents two fragments, one from the 25 S rRNA coding region and the other from the spacer region. The strains were obtained from the following sources: $288C from the Yeast Genetics Stock Center, Berkeley, CA; S. douglasii from Don Hawthorne, Seattle, WA; all other strains from the Centraalbureau voor Schimmelcultur~ Delft, Holland.
DNA probes (e.g., rDNA or the gene for the translation elongation factor ltx24), hybridization signals are seen with DNA of all Saccharomyces species and even other yeast genera. Acknowledgments We thank Sue Klapholz, Jfirg Gafner, and Hans Hegemann for useful comments and Valerie' Lui for typing the manuscript. This work was supported by the Swiss National Science Foundation and by the Deutsche Forschungsgemeinschafl (SFB 272). 24E Schirmaier and P. Philippsen, EMBO Z 3, 3311 (1984).
[1 2] H i g h - E f f i c i e n c y T r a n s f o r m a t i o n Electroporation
of Yeast by
By DA~IF.L M. BECKERand LEONARDGUARE~TE Introduction A prerequisite for molecular biological manipulation of any organism is a reliable and efficient means for introducing exogenous DNA into the cell. Yet each of the techniques in general use for transforming yeast, namely, lithium acetate transformation ~ and spheroplast transformation, 2 suffers from significant limitations. Lithium acetate transformation, although relatively fast and simple, provides only a low efficiency of DNA transfer ( - l0 s colonies//zg of episomal plasmid). Spheroplast transformation, while more efficient ( - 1-5 X 104 colonies//zg), is complicated and time consuming. We present here a method for transforming yeast by electroporation that is extremely simple and an order of magnitude more efficient than spheroplast transformation. A number of groups have attempted previously to introduce macromolecules into yeast by dectroporation, 3-5 but efforts to introduce plasraids have failed to yield transformation efficiencies greater than approxiH. Ito, Y. Fukada, K. Murata, and A. Kimura, J. Bacteriol. 153, 163 (1983). 2 A. Hinnen, J. B. Hicks, and G. R. Fink, Proc. Natl. Acad. Sci. U.S.A. 75, 1929 (1978). 3 H. Hashimoto, H. Morikawa, Y. Yamada, and A. Kimura, Appl. Microbiol. Biotechnol. 21, 336 (1985). 4 I. Karube, E. Tamiya, and H. Matsuoka, FEBSLett. 182, 90 (1985). s I. Uno, K. Fukami, H. Kato, T. Takenawa, and T. Ishikawa, Nature (London) 333, 188
(1988). METHODS IN ENZYMOLOGY, VOL. 194
~ t O 1991 by Academic Pica, Inc. All rightmofrepfoduefion in any form re~rved.