Cloning cell cycle regulatory genes by transcomplementation in yeast

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GENERALMETHODOLOGIES

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residues needed for charging by ESI. This treatment also leads to /3-elimination of cysteine residues, producing a mass change of - 3 4 Da, and conversion of glutamine residues to glutamic acid has been observed ( - 1 Da).

[4] C l o n i n g C e l l C y c l e R e g u l a t o r y G e n e s b y Transcomplementation in Yeast By

CHRIS NORBURY and SERGIO MORENO

Introduction In principle, the identification and molecular cloning of cell cycle regulatory genes can be achieved through a variety of approaches. Cloning strategies based on sequence similarity to previously identified genes, antibody cross-reactivity, or in vitro assays of cell cycle functions combined with biochemical purification have all proved successful. In contrast, genetic approaches offer the possibility of gene isolation through direct selection of defined biological functions in vivo. It would be hard to overestimate the impact of such approaches, particularly those exploiting the power of yeast genetics, on the field of cell cycle regulation. Gene cloning by functional complementation, typically of temperature-sensitive cell cycle mutants, has been achieved in a large number of studies of the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe. In most of these cases, the gene isolated has been derived from the species in which the complementation was performed, but an enduring appeal of the approach lies in its potential for the isolation of cell cycle genes from other, less genetically tractable organisms through transcomplementation in either yeast. The first indication that this could be a feasible strategy came with the cloning of the S. cerevisiae CDC28 gene by complementation of an S. pombe cdc2 's mutant. 1 This pioneering experiment used an S. cerevisiae genomic DNA library constructed in YEP13, a vector designed for use in budding yeast, but which also is maintained episomally after transformation of S. pombe. In many organisms, promoter elements and splicing signals have not been sufficiently well conserved to ensure adequate levels of expression after introduction of genomic DNA fragments into yeast. These difficulties have been circumvented by the use of cDNA libraries, either constructed specifically for the purpose or using heterologous 1 D. Beach, B. Durkacz, and P. Nurse, Nature (London) 300, 706 (1982).

METHODS IN ENZYMOLOGY, VOL. 283

Copyright © 1997by AcademicPress All rightsof reproductionin any form reserved. 0076-6879/97 $25.00

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promoters that retain sufficient function to allow expression of the cloned cDNAs in yeast. In 1987, the full potential of this approach was realized with the cloning by transcomplementation of a human cDNA encoding a functional homolog of the fission yeast cdc2 + gene. 2 A similar study identified a Drosophila cdc2 homolog, 3 and functional homologs of CDC28 were subsequently cloned from human 4'5 and Candida albicans 6 libraries expressed in budding yeast. It is noteworthy that most of the other successful applications of this strategy have also involved complementation of mutations affecting either mitotic regulators, such as weel 7,s and cdc25, 3'9't° or elements required for passage through start, such as the G1 cyclins of budding yeastY~-16 This is not an absolute limitation of the method, however, as indicated by the successful cloning of a human dTMP kinase gene by complementation of a budding yeast cdc8 mutant. 17 An overview of a typical transcomplementation experiment is shown in Fig. 1. Factors Influencing Choice of Library Transcomplementation between different yeast species can be achieved using genomic D N A libraries, 1"6 but in most cases an appropriate cDNA library is required. Libraries suitable for expression in budding or fission yeast have been prepared using cDNA from a variety of human cell

2 M. G. Lee and P. Nurse, Nature (London) 327, 31 (1987). J. Jimenez, L. Alphey, P. Nurse, and D. M. Glover, E M B O J. 9, 3565 (1990). 4 j. Ninomiya Tsuji, S. Nomoto, H. Yasuda, S. I. Reed, and K. Matsumoto, Proc. Natl. Acad. Sci. U.S.A. 88, 9006 (1991). S. J. Elledge and M. R. Spottswood, E M B O J. 10, 2653 (1991). G. Sherlock, A. M. Bahmam A. Mahal, J. C. Shieh, M. Ferreira, and J. Rosamond, Mol. Gen. Gener 245, 716 (1994). 7 M. Igarashi, A. Nagata, S. Jinno, K. Suto, and H. Okayama, Nature (London) 353, 80 (1991). x S. D. Campbell, F. Sprenger, B. A. Edgar, and P. H. O'Farrell, Mol. Biol. Cell 6, 1333 (1995). '~ L. Alphey, J. Jimenez, H. White Cooper, I. Dawson, P. Nurse, and D. M. Glover, Cell 69, 977 (1992). 1{iA. Nagata, M. Igarashi, S. Jinno, K. Suto, and H. Okayama, New Biol. 3, 959 (1991). II M. Dahl, I. Meskiene, L. Bogre, D. T. Ha, I. Swoboda, R. Hubmann, H. Hirt, and E. Heberle Bors, Plant Cell 7, 1847 (1995). 12 E. E. Lahue, A. V. Smith, and T. L. Orr Weaver, Genes Dev. 5, 2166 (1991). 13 p. Leopold and P. H. O'Farrell, Cell 66, 1207 (1991). 14 D. J. Lew, V. Dulic, and S. I. Reed, Cell 66, 1197 (1991). ~ R. Soni, J. P. Carmichael, Z. H. Shah, and J. A. Murray, Plant Cell 7, 85 (1995). J~' A. Koff, F. Cross, A. Fisher, J. Schumacher, K. Leguellec, M. Philippe, and J. M. Roberts, Cell 66, 1217 (1991). 17 j. y . Su and R. A. Sclafani, Nucleic Acids Res. 19, 823 (1991).

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promoter

"N ~K J LEU2"~-"~,--R-Amp

\ ~

Transforminto straiPPr(°.P~i,'.tcdcYe,/Stu.)

Select leu+:cDNAexpression at permissivetemperature

Transferto restrictivetemperature: select leu+, cdc+

Stabilitytest Plasmidrecovery in E. coil Checkcomplementationof yeast defect FIG. l. Overview of a typical transcomplementation experiment. A cDNA library in which cDNA expression is driven by a promoter active in yeast and carries a selectable marker (in this case LEU2) is transformed into the yeast strain to be complemented. A variant technique involves cotransformation of the cDNA library with a second plasmid bearing the selectable marker. After selection for transformants bearing the selectable marker, a period of expression of the cDNA is generally allowed before shift to restrictive conditions to select for cDNAs capable of complementing the yeast defect in question (in this case, a temperature-sensitive cdc mutation). Stability tests are used to distinguish plasmid-dependent complementation from spontaneous chromosomal suppression or reversion mutations. Complementing plasmids are retrieved by transformation of E. coli and subsequently tested for complementing function by a second round of yeast transformation.

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lines,4'5'7"14'16'17 mouse fibroblasts, ~8Xenopus whole ovaries or oocytes, 19-21 and Drosophila embryos or cultured cells. 3"~eA3"22 Successful complementation may be critically dependent on the level of gene expression from the library vector, so choice of promoter is also an important consideration. The SV40 and SP6 heterologous promoters provide constitutive, moderate levels of expression in S. pombe, making possible the use in fission yeast of libraries designed primarily for mammalian or bacterial hosts, either after adaptation by insertion of an arsl-LEU2 fragment3 or by cotransformation with an unlinked selectable markerY Some workers have employed high levels of constitutive expression driven by the adh or ADH (alcohol dehydrogenase) promoter in fission and budding yeast, respectively,12-14a6,2l or by the budding yeast GAP promoter. ~4"17 Perhaps the greatest flexibility is afforded by the use of libraries with regulatable promoters, notably G A L l in S. cerevisiae 4s and nrntl in S. porn be. 18-20.22 Limitations of Technique It is important to appreciate the publication bias in favor of success; in many (mostly anecdotal) cases, attempts to identify functional homologs of yeast cdc genes by transcomplementation have not been fruitful. There are a number of possible explanations for this rather modest success rate. For example, the degree of conservation of protein structure may be insufficient to allow a given gene product to substitute for the analogous yeast protein. The precise minimum degree of conservation required will be gene product dependent, but appears to be lower for enzymatic functions (i.e., the cdc25 family of phosphatases) than it is in cases where the overall tertiary structure of the protein is more critical. Another potential pitfall derives from the fact that appropriate regulation of expression is important for many cell cycle-related genes, as this regulation will usually be lost when the corresponding cDNAs are brought under the control of a heterologous promoter in a library vector. Furthermore, the abundance of a particular cDNA within a library will be determined by the level of expression of the corresponding gene, and longer cDNAs may be underrepresented during library construction for a variety of reasons. It follows that the cDNA sought could be very scarce within the library used, such that prohibitively ~s B. Edgar and C. Norbury, unpublished results (1991). v, D. Patra and W. G. Dunphy, Genes Dev. 10, 1503 (1996). 2o p. B. Carpenter, P. R. Mueller, and W. G. Dunphy, Nature (London) 379, 357 (1996). 2~ j. y . Su and J. L. Maller, Mol. Gen. Gener 246, 387 (1995). 22 K. A. Edwards, R. A. Montague, S. Shepard, B. A. Edgar, R. L. Erikson, and D. P. Kiehart, Proc. Natl. Acad. Sci. U.S.A. 91, 4589 (1994).

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large numbers of transformants would have to be screened; in the worst possible case, the cDNA may not be present at all. In numerous cases, genes isolated by transcomplementation in yeast have been found to have significantly different functions in their respective cells of origin, and in some of these cases have been structurally unrelated to the mutant gene in the strain being complemented. Complementation of G1 cyclin (CLN) defects in S. cerevisiae, for example, has been used to identify a variety of cyclin cDNAs, including those encoding human Aand B-type cyclins, which function during S and G2/M in human cells, TM as well as Drosophila cyclin C and S. pombe pucl ÷, which also may not function as authentic G1 cyclinsJ3'23 Similarly, Xenopus cDNAs isolated on the basis of their ability to suppress the mitotic catastrophe phenotype seen in fission yeast wee1-50, miklA or wee1-50, rad3-136 strains encode proteins unrelated to any of the mutant yeast gene p r o d u c t s 19-21 and a search for cDNAs capable of suppressing a budding yeast mecl mutation identified a human CDC34 homolog. 24Analogous screens using cdc4, cdc15, and S. pombe radl generated cDNAs respectively encoding human cyclin F, Xenopus N-ras, and RACH2, a novel human proteinY -27 This lack of stringency, the extent of which will depend on the nature of the screen being performed, could lead to the identification of physiologically meaningful interactions between gene products, but the potential for selection of false positives should also be kept in mind. Strains Detailed methods for budding and fission yeast manipulations can be found elsewhere. 2s Here we describe briefly a few very basic techniques. Storage of Yeast Strains

Long-Term Storage The best way to keep yeast strains is to store them frozen at - 7 0 ° in media containing 25% (v/v) glycerol. Yeast cells are viable for many years under these conditions. 23 S. L. Forsburg and P. Nurse, Nature (London) 351, 245 (1991). 24 S. E. Plon, K. A. Leppig, H. N. Do, and M. Groudine, Proc. Natl. Acad. Sci. U.S.A. 9@, 10484 (1993). 25 C. Bai, R. Richman, and S. J. Elledge, E M B O J. 13, 6087 (1994). z6 W. Spevak, B. D. Keiper, C. Stratowa, and M. J. Castanon, Mol. Cell. Biol. 13, 4953 (1993). 27 S. Davey and D. Beach, Mol. Biol. Cell. 6, 1411 (1995). 28 C. Guthrie and G. R. Fink (eds.), Methods Enzymol. 194 (1991).

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1. Grow cells in 0.8 ml Y E P D medium for S. cerevisiae (YES for S. pombe; see Tables I and II) to early stationary phase (OD600 = 2) at 25-32 ° for 1-2 days. 2. Mix with 0.8 ml of sterile 50% glycerol in a cryotube. Store the cryotube at - 7 0 °.

Short-Term Storage For short-term storage cells can be kept on plates at 4 ° for about 1 month. Strains do not store well on minimal medium or, in the case of S. pombe, in phloxine B-supplemented medium. Cells should be patched out on a fresh Y E P D or YES agar plate overnight before use. Waking u p S t r a i n s For strains stored on glycerol at -70°: 1. Using a sterile toothpick, scrape off a small amount of frozen glycerol stock and patch onto a Y E P D plate for S. cerev&iae (YES for S. pombe), keeping the stock frozen. If necessary, put the cryotube in dry ice to prevent defrosting of the stock. 2. Incubate at 25-32 ° for 2-5 days, depending on the strain. 3. When growth is visible, streak out to single colonies onto a Y E P D plate for S. cerev&iae (YES for S. pombe) and incubate at 25-32 ° for 2-3 days. Strains stored as patches on plates at 4 ° are streaked out onto a fresh Y E P D or YES plates directly, and incubated at 25-32 ° as appropriate. It is essential to check the phenotype of any reisolated strain before carrying out any experiments. Routine tests should include temperature sensitivity or cold sensitivity, auxotrophic markers, ploidy, and mating type. G r o w t h Media for Saccharomyces cerevisiae a n d

Schizosaccharomyces pombe We usually prepare 4 liters of yeast medium at a time by dissolving the components in water using a 5-liter flask. The medium is then distributed into 10 500-ml Duran bottles (Schott Glaswerke, Mainz, Germany), each containing 400 ml of medium and autoclaved with the caps loose at 10 psi for 20 rain. At this pressure, very little caramelization of glucose takes place. We usually autoclave everything together, but if caramelization is a problem, glucose or galactose can be sterilized independently as a 20% stock and added to the media when it has cooled. Solid medium is made

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by adding 10 g of Bacto-agar (2.5% final concentration) to each Duran bottle before autoclaving. Plates may be prepared immediately by allowing the medium to cool down for 30-60 min in a water bath at 53 ° and then pouring it into plates (16-18 plates per bottle). Alternatively, solid medium may be kept in Duran bottles for months and microwaved (one or more bottles) at medium power for 10-20 min just before use. Budding yeast minimal medium is prepared with all the supplements added. Minimal medium for selection (also called "drop outs") is prepared with all the supplements added except the one that is going to be selected (i.e., -Leu, -Ura, etc.). Fission yeast minimal medium is sterilized without supplements and the required supplements are added after sterilization from sterile stock solutions. Phloxine B and thiamin are also added from filter-sterilized stocks when the medium has cooled down. Common rich and minimal media for S. cerevisiae and S. pombe are described in Tables I and II. A more detailed description of media and growth conditions can be found elsewhere} 9'3° We usually buy yeast extract, peptone, and agar from Difco laboratories (Detroit, MI) and supplements, phloxine B, thiamin, and galactose from Sigma (St. Louis, MO). Alternatively, Bio]01, Inc. (Vista, CA), sells yeast media ready to dissolve in water and to autoclave. Transformations Here we describe two protocols for yeast transformation. The first is a lithium acetate transformation protocol that we obtained from Steve Elledge's laboratory (Baylor College of Medicine, Houston, TX). This protocol works very well for both S. cerevisiae and for S. pombe and gives reproducibly high transformation frequencies. As an alternative, we include an electroporation protocol that is quick, convenient, and can give even higher transformation frequencies, but is not always as reproducible as the lithium acetate protocol. We tend to use lithium acetate for transformation with cDNA libraries and electroporation with genes already cloned in yeast vectors. Maximal transformation efficiency depends mainly on collecting cells at the right density and using good quality DNA (CsC1- or columnpurified D N A gives the best efficiencies). These are some guidelines for successful transformations using cDNA libraries. 1. Plan the experiment several days ahead of time. Make sure that all the media, plates, and solutions are made or will be made by the day of the transformation. 29 S. Moreno, A. Klar, and P. Nurse, Methods Enzymol. 194, 795 (1991). 30 F. Sherman, Methods Enzymol. 194, 3 (1991).

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TABLE I

Saccharomyces cerevisiae MZD1A" Medium Complex YEPD (for routine growth)

YEPGal

Synthetic SC (synthetic complete media)

Component Yeast extract Peptone Glucos& Deionized water to 1 liter Yeast extract Peptone Galactose t' Deionized water to 1 liter Yeast nitrogen base (without amino acids) Glucose Supplement powder Leu Ade PABA Ala, Arg, Asn, Asp, Cys, Gin, Glu, Gly, His, Ile, Lys, Met, Phe, Pro Ser, Thr, Trp. Tyr, Val, and Ura Prepare the following solutions, autoclave and mix 400 ml deionized water + 2 g of the supplement powder + 20 g agar 560 ml deionized water + 6.7 g of YNB (without amino acids) 40 ml 50% glucose (20 g glucose + 30 ml deionized water) Prepare as above, but use supplement mix lacking amino acid or nitrogen base that is going to be selected f

Supplement powder

SC (solid media) (1 liter)

Selection

Amount (g/liter) 10 20 20 10 20 25

7 20 2 0.5 4 0.2 2 each

"These media can also be purchased from Biol01, Inc. i, To avoid caramelization of the sugar, glucose and galactose are sterilized separately as 20% stocks in water and then added to the autoclaved medium. 2. A l w a y s start the e x p e r i m e n t by i n o c u l a t i n g cells into 2 0 - 5 0 ml of c o m p l e x m e d i u m , Y E P D for S. cerevisiae a n d Y E S for S. pombe, with a fresh single c h e c k e d colony. G r o w this i n o c u l u m in a s h a k i n g w a t e r b a t h or air s h a k e r for 1 or 2 days at 2 5 - 3 2 °, d e p e n d i n g o n the strain. 3. H a r v e s t the cells at the right optical density. L o o k at the cells u n d e r the m i c r o s c o p e to m a k e sure they are h e a l t h y a n d free from bacterial contamination.

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GENERAL METHODOLOGIES T A B L E II

Schizosaccharomyces pombe MEDIA" Medium Complex YES

Minimal EMM

Component

Amount (g/liter)

Yeast extract Glucose Adenine, histidine, leucine, uracil, and lysine Deionized water to 1 liter

5 30 50 mg each

Potassium hydrogen phthalate Na2HPO4 NH4CI Glucose Salts (50×) Vitamins (1,000×) Minerals (10,000×)

3 2.2 5 20 20 ml 1 ml 0.1 ml

Salts (50× stock) MgC12 • 6H20 CaCI2 • 2H20 KC1 Na2SO4 A d d deionized water to 1 liter; autoclave and store at 4 ° Vitamins (1,000× stock) Calcium pantothenate Nicotinic acid

myo-Inositol Biotin A d d deionized water to 1 liter; autoclave and store at 4 ° Minerals (10,000× stock) H3BO3 MnSO4 ZnSO4 •7H20

FeC13 • 6H:O Molybdic acid KI CuSO 4 •5H20

Citric acid A d d deionized water to 1 liter; autoclave and store at 4 °

53.5 0.74 50 2

1 10 10 10 mg

5 4 4 2 2 1 0.4 10

Individual stocks of adenine, leucine, and histidine are prepared at 7.5 g/liter and uracil at 3.75 g/liter in deionized water. Autoclave in 200-ml aliquots and add to autoclaved media to a final concentration of 225 mg/liter. Histidine should be protected from light. Thiamin stock (4,000×) is made in water at 20 mg/ml, filter sterilized, and kept at room temperature wrapped in aluminum foil. Thiamin is added to the media below 60 °. Phloxine B stock (1,000×) is made in water at 5 mg/ml, filter sterilized, and kept at room temperature wrapped in aluminum foil. Phloxine B is added to the media below 60 °. "These media can also be purchased from Biol01, Inc.

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4. Fungal contamination may occur at any point during the experiment. To avoid plate surgery later, use only material that has been sterilized and clean the work area carefully with ethanol. D N A contamination is another common problem (sometimes the yeast gene is reisolated when screening mouse cDNA libraries!). To prevent this, avoid centrifuge tubes, micropipettes, and solutions that might be contaminated with plasmid DNA. Use disposable centrifuge tubes and solutions freshly made for each experiment.

Lithium Acetate Procedure 1. From a single checked colony, grow a 20-ml culture to stationary phase in YEPD (S. cerevisiae) or YES (S. pombe) with shaking at 30° [25 ° for ts (temperature-sensitive) mutants] for 24-48 hr. 2. Use this culture to inoculate a 500-ml culture of YEPD (S. cerevisiae) or YES (S. pombe), shaking overnight at 30 ° (25 ° for ts mutants) to o n e 0 D 6 0 0 = 1. 3. Harvest the cells at 3000 rpm for 3 min. 4. Wash twice in 100 ml of sterile distilled water and once with 50 ml of LiSorb (100 mM lithium acetate, 1 mM E D T A , 1 M sorbitol, 10 mM Tris-HC1, pH 8.0). Resuspend in 50 ml of LiSorb and incubate at 30 ° for 20 min, shaking. 5. Spin down as above and resuspend in 625 Ixl LiSorb. Hold on ice. 6. Prepare carrier D N A mix. a. Boil 200 ~1 (20 mg/ml) sheared salmon sperm D N A for 7-10 rain. b. Cool the D N A to room temperature. c. Add 800/xl LiSorb at room temperature and mix by pipetting up and down. d. Add 40 p,g library DNA. 7. Add 100/xl of the above mix to 100 tzl of cells from step 5. 8. Incubate 10 min at 30 °. 9. To 100/xl cells + D N A add 900 txl 40% polyethylene glycol (PEG) 3500 in 100 mM LiAcTE (100 m M lithium acetate, 10 mM Tris, pH 8, 1 mM EDTA) and incubate at 30 ° for 30 min. Heat shock at 42 ° for 7 rain. 10. Pool the cells and add to 100 ml minimal selective liquid media, shake at 30 ° (25 ° for ts mutants) for 1-5 hr. Harvest the cells and resuspend in 6 ml of minimal selective liquid media and plate 100 txl per 9-cm plate (300 txl per 15-cm plate). 11. Colonies grow after about 3-5 days. Efficiency should be between 5 x

10 4

and 10s colonies//xg library DNA.

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Electroporation For this protocoP 1 it is important to prechill tubes, electroporation cuvettes, water, and 1 M sorbitol and to keep the cells on ice all the time. Cells are carefully washed with sterile water and 1 M sorbitol to remove extracellular ions.

Schizosaccharomyces pombe Protocol 1. From a single colony, grow a 20-ml culture to early stationary phase in YES medium with shaking at 30° (25 ° for ts mutants) for 24-48 hr. 2. Use this culture to inoculate 100 ml culture to a density of 1 x 107 cells/ml (OD595 = 0.5) in minimal medium. Transformation frequency is not harmed by growth until early stationary phase (OD595 = 1.5). 3. Harvest ceils by spinning at 3000 rpm for 3 rain at 4° in prechilled 50-ml Falcon tubes (Becton-Dickinson Labware, Franklin Lakes, N J). Wash once by resuspending in 50 ml ice-cold water and harvesting; a second time by resuspending in 30 ml ice-cold 1 M sorbitol and harvesting. 4. Resuspend cells in ice-cold 1 M sorbitol at a density of 1-5 × 109 cells/ml. 5. Add 40-100 /xl of the cell suspension to chilled Eppendorf tubes containing the DNA for transformation (100 ng) and incubate on ice for 5 rain. The DNA should be in water or in TE (10 mM TrisHC1, 1 mM EDTA, pH 8.0) and must be in as small a volume as possible. 6. Set the electroporator (Bio-Rad, Richmond, CA) to 1.5 kV, 200 11, 25/zF. 7. Transfer cells and DNA to a prechilled 0.2-cm sterile electroporation cuvette. Tap to the bottom, dry the cuvette with tissue paper, and pulse it. Add 0.9 ml of ice-cold 1 M sorbitol immediately to the cuvette and mix. 8. Plate cells as soon as possible onto S. pombe minimal medium. Transformants appear in 4-6 days at 30°. Transformation efficiencies of >105//zg DNA are expected. Maximal efficiency (transformants//zg) will be obtained with <10 ng of DNA; the largest number of transformants will be obtained with 100 ng of DNA. Using more than 100 ng or including carrier DNA decreases both the efficiency and the total number of transformants. 31D. M. Becker and L. Guarente, Methods EnzymoL 194, 182 (1991).

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Saccharomyces cerevisiae Protocol 1. From a single colony, grow a 20-ml culture to early stationary phase in Y E P D medium with shaking at 30 ° (25 ° for ts mutants) for 24-48 hr. 2. Use this culture to inoculate 150 ml culture ceils in Y E P D to OD600 = 1.2-1.5. 3. Harvest ceils by spinning at 3000 rpm for 5 min at 4 ° in prechilled 50-ml Falcon tubes (Becton-Dickinson Labware, Franklin Lakes, NJ). Wash once by resuspending in 50 ml ice-cold water and harvesting; a second time by resuspending in 30 ml ice-cold 1 M sorbitol and harvesting. 4. Resuspend cells in 0.5 ml ice-cold 1 M sorbitol. 5. Add 40-100 /xl of the cell suspension to chilled Eppendorf tubes containing the D N A for transformation (100 ng) and incubate on ice for 5 min. The D N A should be in water or in T E (10 mM TrisHC1, 1 mM E D T A , pH 8.0) and must be in as small a volume as possible. 6. Set the electroporator (Bio-Rad) to 1.5 kV, 200 tl, 25/xF. 7. Transfer cells and D N A to a prechilled 0.2-cm sterile electroporation cuvette. Tap to the bottom, dry the cuvette with tissue paper, and pulse it. Immediately add 0.9 ml of ice-cold 1 M sorbitol to the cuvette and mix. 8. Plate cells as soon as possible onto S. cerevisiae minimal medium containing 1 M sorbitol. Transformants should appear within 4-6 days at 30 °. The transformation frequency should be 2-5 × 105 transformants//xg of DNA. Selection Protocols Deciding the right conditions for selection after transformation can take some time. Pilot experiments should be undertaken to arrive at a protocol that is likely to work with the yeast strain being used. It may be necessary to optimize conditions to obtain a high number of transformants and the right number of transformants per plate (aim for 2000 to 5000 transformants per 9-cm petri dish) by using different amounts of D N A in the transformation protocol or plating several dilutions of the final mix. Some strains do not transform very well, especially in S. cerevisiae. Transformation proficiency can sometimes be improved by backcrossing with a strain known to be more competent for transformation.

Complementation of Temperature-Sensitive Mutants When using a temperature-sensitive (ts) or a cold-sensitive (cs) mutant, it is worth determining the exact temperature at which the cells cease to

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grow. Increase (for ts mutants) or decrease (for cs mutants) this temperature by one or two degrees for the selection of positive clones. If the yeast gene that complements the mutant has already been cloned, it is useful to check that it complements the phenotype at the chosen temperature. In the classical way of cloning genes by complementation, transformants are grown at the permissive temperature in selective minimal media and then replica plated to the restrictive temperature. Plates are screened visually every day for 2-4 days for colonies that grow at this temperature. In the case of fission yeast, it is useful to add phloxine B to the plates to help identify the positive pink colonies in a background of dark red negative colonies. This procedure usually works very well for genomic libraries because the number of colonies to be screened is relatively small, between 20,000 and 50,000 colonies (10 to 25 plates at 2000 colonies per plate). For cDNA libraries, the number of clones to be screened is usually 10 or 20 times higher, and it is probably a better idea to select directly at the restrictive temperature. For this, cells are plated at the permissive temperature for 12-24 hr and then shifted to the restrictive temperature until colonies appear. This approach allows some time for cDNA expression before selection begins and is especially important when using libraries with regulatable promoters such as G A L l (induced in galactose) in S. cerevisiae or n m t l (repressed by thiamin) in S. pombe. Cells should be grown for 8-10 hr on galactose plates for G A L l and 20-24 hr in minimal medium without thiamin for the n m t l promoter. The fission yeast n m t l promoter produces a significant level of expression, even when it is repressed, such that the human CDC2 cDNA can fully rescue a cdc2 t" mutant when expressed from the n m t l promoter in the presence of thiamin. Other genes need to be expressed at a higher level to be able to complement. For this reason, when using libraries containing the n m t l promoter, it is worth trying complementation both in the presence (low-level expression) and in the absence (high-level expression) of thiamin. In the first case, cells are plated in minimal medium containing 5 tzg/ml thiamin and, in the second, no thiamin is added to the medium. It takes 15-20 hr for this promoter to be derepressed, which means that cells need to be maintained at the permissive temperature for this period before shifting. Stability Test This test is useful for distinguishing between a chromosomal suppressing mutation (a reversion or gene conversion event that may have occurred during transformation or selection) and the presence of a suppressing plasmid-borne cDNA. In the first case, the phenotype is maintained after relaxing selection for the plasmid marker and, in the second case, the plasmid

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will be lost in the absence of selection and with it the complementation phenotype. The procedure is as follows: 1. Streak the transformant out to single colonies on YEPD agar (YES agar for S. pombe) with no selection for about 3 days until colonies form. 2. Replica plate to selective medium (e.g., 35 ° on YES for a ts strain, 25 ° on minimal for the auxotrophic marker), and check for the stability of the prototrophic and/or other plasmid-borne phenotypes. When using a regulatable promoter, it may be possible to check if complementation depends on promoter activity. This is done by replica plating the colonies to minimal medium with glucose or galactose, for the G A L l promoter, or medium with or without thiamin for the nmtl promoter.

Plasmid Rescue

Recovering Plasmids from Schizosaccharomyces pombe Recovering ars-less plasmids from S. pombe is difficult, as they often seem to form multimers, and rearrangements (tandem duplication or deletions) are frequent, In contrast, plasmids containing an ars do not tend to rearrange and are easy to recover. It is worth noting also that yeast material in the final preparation appears to inhibit Escherichia coli transformation and thus using more of the preparation may not be a good idea. To recover a plasmid from an S. pombe transformant, two procedures are routinely used:

Protocol A 1. Grow up 10 rnl of cells under selective conditions to OC59~ = 1 (2 x 107 cells/ml). 2. Spin down the cells at 3000 rpm and 4 ° for 5 rain. 3. Resuspend in 1.5 ml of sterile 50 mM citric acid, 50 mM NazHPO4, 1.2 M sorbitol, pH 5.6. Add fresh Zymolyase-20T (2 mg/ml, final concentration). Transfer to an Eppendorf tube and incubate at 37 ° for 1 hr. 4. Pellet the cells at 13,000 rpm and 4 ° for 30 sec. Resuspend in 300 /zl TE. 5. Add 35/~110% SDS and mix. The yeast cells should lyse at this point. 6. Add 100 tzl 5 M potassium acetate, mix and leave on ice for 30 min. 7. Spin down at 13,000 rpm and 4 ° for 10 min. 8. Add 50 tzl of supernatant to 100/zl NaI solution (Geneclean Kit, Biol01, Inc.) with 5 tzl glass milk (Geneclean Kit, Biol01, Inc.).

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[4]

9. Incubate for 5 min at room temperature. 10. Spin at 13,000 rpm for 5 sec at room temperature. Discard the supernatant and wash the pellet three times with 400/xl of ice-cold NEW wash (Geneclean Kit, Biol01, Inc.). 11. Elute the DNA with 20/zl of TE at 55 ° for 3 min. 12. Spin out the glass milk and keep the supernatant. 13. Transform 5/xl of the supernatant into 100/xl of competent E. coli JA226 cells. The use of the Geneclean kit improves the transformation frequency by at least 10-fold; also it is very important to use a recBC E. coli strain such as JA226 when using 2/xm or ars-less plasmids. For arsl-based plasmids recA strains, such as E. coli, DH5 can be used.

Protocol B 1. Grow small cultures (at least 1.4 ml) with selection. 2. Collect the cells by a 5-sec spin in a microfuge at 13,000 rpm. 3. Discard the supernatant and briefly vortex the tube to resuspend the pellet in the residual medium. 4. Add 0.2 ml of 2% (v/v) Triton X-100, 1% SDS, 100 mM NaC1, 1 mM EDTA, 10 mM Tris-HC1, pH 8.0. 5. Add 0.2 ml phenol: chloroform : isoamyl alcohol (25 : 24 : 1, v/v) and 0.3 g acid-washed glass beads (Sigma). 6. Vortex for 2 min and then microfuge at 13,000 rpm for 5 min. 7. Transfer the upper aqueous layer to a fresh Eppendorf tube and extract with 200 tzl phenol : chloroform : IAA (25 : 24 : 1, v/v). 8. Precipitate the DNA, wash with 70% (v/v) ethanol, dry, and resuspend in 10/zl TE. 9. Use 1-5/~l for transformations of competent E. coli.

Recovering Plasmids from Saccharomyces cerevisiae Start with a fresh patch on a plate. 1. Resuspend one toothpick of yeast cells in 100/zl of 100 mM NaC1, 0.5% SDS, 1 mM EDTA, 100 mM Tris-HC1, pH 9.0, in an Eppendoff tube. 2. Add 100/xl of acid-washed glass beads (Sigma). Vortex 20 sec twice. Cool on ice. 3. Add 100/xl of phenol:chloroform (50:50, v/v). Vortex 5 sec. Spin down 4 min at 13,000 rpm and 4 °. Take 50/zl of the aqueous phase and transfer to a fresh sterile Eppendorf tube.

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SORTING OF TRANSFECTED

CELLS

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4. Add 50/xl of phenol : chloroform. Vortex 5 sec. Spin down 4 min at 13,000 rpm. Take 30/xl of the aqueous phase and transfer to a fresh sterile Eppendorf tube. 5. Add 30 txl of phenol:chloroform (50:50, v/v). Vortex 5 sec. Spin down at 13,000 rpm for 4 min. Take 20/xl of the aqueous phase and transfer to a fresh sterile Eppendorf tube. Note: Avoid any interphase in each phenol extraction. 6. Add 2.5 volumes of ethanol (no salts). Spin down 5 min at 13,000 rpm and 4°. Wash the pellet with 0.5 ml of 70% ethanol and dry under vacuum. 7. Resuspend the pellet in 50/xl of sterile water or TE. 8. Use 10/xl to transform E. coli DH5, JA226, or XL1-Blue. The recovered plasmids could be digested with several restriction enzymes to check how many different inserts have been cloned. They should then be reintroduced into yeast to show complementation on retransformation. Acknowledgments We thank Carlos Vazquez for comments on the manuscript. Research in our laboratories is supported by the Imperial Cancer Research Fund, the Human Frontier Science Program. the Ramdn Areces Foundation, and the CICYT.

[5] F l u o r e s c e n c e - A c t i v a t e d C e l l S o r t i n g o f Transfected Cells

By PETER D. ADAMS, PETER LOPEZ, WILLIAM R. SELLERS, a n d WILLIAM G. KAELIN, JR.

Introduction Fluorescence-activated cell sorting (FACS) is a technique that can measure the fluorescence intensity of individual cells within a population. The cells within a population are labeled with a fluorescent label (fluorochrome) in such a way that the intensity of fluorescence of an individual cell provides a quantitative measure of the particular parameter of interest. By staining cells with fluorochromes of different emission wavelengths, multiple parameters can be measured simultaneously. If necessary, FACS can be used to physically separate the cells according to the particular parameters being measured (see below).

METHODS IN ENZYMOLOGY, VOL. 283

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