Isolation and Identification of Yeasts from Natural ... - Library Science

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48 4. Notes Curran and Bugeja 1. This protocol is written assuming that the parent strains carry complemen- tary auxotrophic markers such that hybrid fusants are prototrophic. For parent strains that share an auxotrophic deficiency, the selective minimal medium must, of course, be appropriately supplemented. 2. Protoplasts from different strains can vary in their osmotic sensitivity, but 0.8 to l.UMsorbitol is adequate for most S. cerevisiae strains. 3. The fusion efficiency of different PEG batches varies considerably. If the fusion efficiency is low but regeneration is adequate, try different sources of PEG. 4. This particular combination of enzymes and j3-mercaptoethanol works extremely well, but any enzyme or combination of enzymes which pro- duce yeast protoplasts can be used, e.g., Zymolase, Glusulase, and so on. 5. The speed at which different strains protoplast varies to some extent, but the most important aspect of protoplasting is the physiological state of the cells. Early to midexponentially growing cells are the most suitable. If there is difficulty in protoplasting, use younger cells. 6. It is of vital importance to embed the protoplasts because yeast protoplasts (unlike other fungi) cannot regenerate on the surface of the agar. 7. Fusion and regeneration frequencies vary considerably from strain to strain. 8. The transfer of cytoplasmic markers is extremely efficient during protoplast fusion, but 3% glycerol replaces the 2% glucose in the regeneration medium when wild-type and petite mutants are fused (3). 9. The percent of fused protoplasts that can undergo karyogamy appears to be restricted to protoplasts containing nuclei at a specific point in Gl of the cell cycle (4). Thus, when asynchronous populations of cells are used in a fusion, the percentage of fused protoplasts that undergo karyogamy is very low. Procedures that increase the proportion of cells at this point in Gl greatly improve hybrid formation frequencies (4). The most efficient means of preparing a population of cells at this point in Gl is to treat Mat a cells with 5 U of a factor (Sigma, St. Louis, MO) for 3-4 h before protoplasting them (5). This can increase the fre- quency of hybrid formation 20-fold (depending on the strains used). A similar procedure using factor a on Mat a cells is not as efficient (6). Acknowledgments The authors wish to thank Gail Cohen, Ruth Graham, and Jean Smith for preparing this manuscript.
Protoplasts in S. cerevisiae 49 References 1 Maggio, B., Ahkong, Q. F , and Lucy, J. A (1976) Poly(ethylene glycol) surface potential and cell fusion. Bzochem J 158,647-650. 2 PapahadJopoulos, D , Vail, W J , Pangborn, W. A, and Poste, G (1976) Studtes on membrane fusion II Induction of fusion m pure phospholtptd membranes by calcium ions and other dwalent metals Blochem Blophys Acta 448,265-283 3 Curran, B P G and Carter, B. L. A. (1986) o-factor enhancement of hybrid formation by protoplast fusion in the yeast S cerevwzae II Curr Genet. 10,943-945 4. Curran, B. P. G. and Carter, B L A (1989) The a-factor enhancement of hybrid formatron by protoplast fusion in S cerevwae can be mimtcked by other procedures Curr Genet 15,303-305. 5. Curran, B. P. G. and Carter, B. L. A. (1983) a-factor enhancement of hybrid formation by protoplast fusion m the yeast S cerevwae J Gen Mlcroblol 129, 1539-1591 6. BugeJa, U. C., Whittaker, P A., and Cut-ran, B. P G. (1988) The effect of a-factor on hybrid formation by protoplast fusion in S cerevisiae. Ferns Mcrob Letts 51, 101-104.
Page 1 and 2: CHAPTER 1 Isolation and Identificat
Page 3 and 4: Identification of Yeasts 3 vegetati
Page 5 and 6: CHAPTER 2 Maintenance and Culture o
Page 7 and 8: Maintenance and Culture of Yeasts 7
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Page 36 and 37: 36 Spencer and Spencer 18. Mutants
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Page 44 and 45: 44 Spencer and Spencer 6. Cytoducti
Page 46 and 47: 46 Curran and Bugeja The fused prot
Page 51 and 52: CHAPTER 6 Meiotic Analysis John F.
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Page 55 and 56: Meiotic Analysis 55 3. Shake the su
Page 57 and 58: Meiotic Analysis 57 Temperature for
Page 59 and 60: CHAPTER 7 Ascus Dissection Carl Sau
Page 61 and 62: Ascus Dissection 61 Fig 1. The Sing
Page 63 and 64: Ascus Dissection 63 on the needle,
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Page 80 and 81: 80 Bignell and Evans ST’3), as we
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98 Vaughan-Martini and Martini 4. C
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100 Vaughan-Martini and Martini 18.
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102 Vaughan-Martini and Martini 6.
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104 Section 3.1. is a straightforwa
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106 3.2. Small-Scale Isolation of S
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CHAPTER 12 Isolation of Mitochondri
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Isolation of Mitochondrial DNA 111
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Isolation of Mitochondrial DNA 113
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Isolation of Mitochondrzal DNA 115
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CHAPTER 13 Isolation of Yeast Plasm
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Isolation of Yeast Plasma Membranes
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Isolation of Yeast Plasma Membranes
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124 Quail and Kelly HO Fig 1. The s
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126 Quail and Kelly 3. Methods 3.1.
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Table 2 Mass Fragmentation Patterns
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130 Quail and Kelly e CONTROL TREAT
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CHAPTER 15 Peroxisome Isolation Ben
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Peroxisome Isolation 135 ODhOa of 1
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Peroxisome Isolation 5 lb 20 Fractt
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CHAPTER 16 Transformation of Lithiu
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Transformation of Yeast 141 2. YEPD
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Transformation of Yeast 143 3.3.2.
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Transformation of Yeast 145 12 Grit
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148 Johnston and DeVit The biolisti
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150 Johnston and DeVit Macrocarrier
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Johnston and DeVit 5. The sample pl
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CHAPTER 18 Genomic Yeast DNA Clone
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Genomic Yeast DNA Clone Banks 157 e
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Genomic Yeast DNA Clone Banks 159 2
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Genomic Yeast DNA Clone Banks 161 f
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Genomic Yeast DNA Clone Banks 177 n
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Genomic Yeast DNA Clone Banks 179 I
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Genomic Yeast DNA Clone Banks 181 f
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190 Kleinman 3. Petri dishes with a
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192 Kleinman Reference 1 Grunstem,
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194 Jordan, Mount, and Hadfield pla
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196 Jordan, Mount, and Hadfield DNA
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198 Jordan, Mount, and Hadfield mar
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200 Jordan, Mount, and Hadfield c.
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202 Jordan, Mount, and Hadfield 4.
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CHAPTER 21 Determination of Chromos
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Determination of Chromosome Ploidy
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CHAPTER 22 Chromosome Engineering i
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Chromosome Engineering in Yeast 219
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Garfinkel solutions of 20% [w/v] an
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230 4. Notes Garfinkel 1. An effect
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232 Garfinkel typic features. In ms
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234 Garfihkel YFQ HIS3 Tetrad or ra
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Garfinkel There are several ways to
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CHAPTER 24 Reporter Gene Systems fo
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Reporter Gene Systems 241 either a
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Reporter Gene Systems 243 3.1. Solu
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Reporter Gene Systems 245 0.5 - 0.4
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Reporter Gene Systems 247 may be ch
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CHAPTER 25 Preparation and Use of Y
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Cell-Free Translation Systems 251 5
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260 Grant, Fitch, and Tuite Fig. 1.
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262 Grant, Fitch, and Tuite 2. Mate
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264 Grant, Fitch, and Tuite 3.2. SD
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266 Grant, Fitch, and Tuite 9. Run
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CHAPTER 27 Preparation of Total RNA
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Preparation of Total RNA 271 satura
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Preparation of Total RNA 273 N--- A
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Preparation of Total RNA 275 3. Soa
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CHAPTER 28 mRNA Abundance and Half-
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mRNA Abundance and Half-Life Measur
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298 Sagliocco, Moore, and Brown The
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300 Sagliocco, Moore, and Brown m a
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302 Sagliocco, Moore, and Brown PUM
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304 Sagliocco, Moore, and Brown 1 1
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306 Sagliocco, Moore, and Brown A C
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308 Sagliocco, Moore, and Brown 2 U
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310 Sagliocco, Moore, and Brown Abs
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CHAPTER 30 Induction of Heat Shock
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Heat Shock Proteins and Thermotoler
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Heat Shock Proteins and Thermotoler
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Vondrejs and Palkovci It should be
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322 Vondrejs and PalkovcE II t I/,
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324 Vondrejs and Palkovci 9. The co
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Palkovci and Vondrejs disadvantage
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Palkov& and Vondrejs Fig. 1. Nystat
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CHAPTER 33 Application of Killer To
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Application of Killer Toxins 333 2.
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Application of Killer Toxins 335 2.
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Application of Killer Toxins 337 gr
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CHAPTER 34 Killer Plaque Technique
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Killer Plaque Technique for Protopl
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CHAPTER 35 Genotoxicity Testing in
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Genotoxicity Testing in Sch. pombe
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356 Stansfield and Kelly by binding
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358 Stansfield and Kelly 450 wavele
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Stansfield and Kelly 11. Monitor th
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362 Stunsfield and Kelly 450 wavele
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12 13 14 15. 16. Stansfield and Kel
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366 Stansfield and Kelly References
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368 Ashby and Beezer prmciple where
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370 Ashby and Beezer 10 Power/micro
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372 Ashby and Beezer c Solution C,
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374 Ashby and Beezer 11. When a bas
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376 Ashby and Beezer 10 Power/mlcro
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378 Ashby and Beezer 8 9 10 A speci
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380 Ashby and Beezer 0 2 4 6 6 10 1
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382 Ashby and Beexer 11 Jo&n, N. (1
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384 StreiblovcE and Hagek Basically
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386 Streiblovb and HaSek 3.2. Vital
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388 StreiblovcE and HaSek Sudan sta
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390 Streiblov& and HaBek 2. Fixatio
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392 HaSek and Streiblovb Tinopal LP
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394 HaSek and Streiblovci 7. Calcof
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396 Ha5ek and StreiblovcE 3.1.2. Fl
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398 Hagek and Streiblovh Fig. 1. Ep
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400 HaSek and Streiblov& 4. Incubat
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Ha%ek and Str beiblovd Fig. 2. Epif
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404 HaSek and Streiblov6 9 When the
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CHAPTER 40 Immunoelectron Microscop
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Immunoelectron Microscopy 409 techn
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Immunoelectron Microscopy 411 The f
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Immunoelectron Microscopy 413 3. Me
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Immunoelectron Microscopy 415 4. Ta
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Immunoelectron Microscopy 417 have
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Immunoelectron Microscopy 419 to be
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Immunoelectron Microscopy 421 5. va
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