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The art and design of genetic screens: yeast

Key Points

  • Classical yeast genetics offers unique tools for discovering gene function in two evolutionarily diverse unicellular organisms: the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe.

  • The tools of classical genetics provide complementary strategies to take advantage of completed genome sequences.

  • Genetic approaches begin with isolation of mutants that affect the process of interest.

  • Synthetic enhancement, including suppression and synthetic lethality screens, allow definition of genetic networks starting from a single mutant allele.

  • Genetic interactions also lead to testable predictions about physical interactions.

  • Plasmid-based screens and functional tests facilitate the characterization of previously cloned genes and the isolation of novel alleles.

Abstract

Understanding the biology of complex systems is facilitated by comparing them with simpler organisms. Budding and fission yeasts provide ideal model systems for eukaryotic cell biology. Although they differ from one another in terms of a range of features, these yeasts share powerful genetic and genomic tools. Classical yeast genetics remains an essential element in discovering and characterizing the genes that make up a eukaryotic cell.

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Figure 1: Morphology of the two yeasts.
Figure 2: Examples of mutant phenotypes in studies of cell-cycle and chromosome dynamics.
Figure 3: Suppressor mechanisms.
Figure 4: Cloning suppressors.
Figure 5: Synthetic lethal screens.
Figure 6: Plasmid shuffle.

References

  1. 1

    Goffeau, A. et al. Life with 6000 genes. Science 274, 546–567 (1996).

    CAS  Article  Google Scholar 

  2. 2

    Beggs, J. D. Transformation of yeast by a replicating hybrid plasmid. Nature 275, 104–109 (1978).

    CAS  Article  Google Scholar 

  3. 3

    Rothstein, R. One step gene disruption in yeast. Methods Enzymol. 101, 202–211 (1983).

    CAS  Article  Google Scholar 

  4. 4

    Oliver, S. G. From gene to screen with yeast. Curr. Opin. Genet. Dev. 7, 405–409 (1997).

    CAS  Article  Google Scholar 

  5. 5

    Oliver, S. G., Winson, M. K., Kell, D. B. & Banganz, F. Systematic functional analysis of the yeast genome. Trends Biotechnol. 16, 373–378 (1998).

    CAS  Article  Google Scholar 

  6. 6

    Sipiczki, M. Phylogenesis of fission yeasts — contradictions surrounding the origin of a century old genus. Antonie Van Leeuwenhoek 68, 119–149 (1995).

    CAS  Article  Google Scholar 

  7. 7

    Paquin, B. et al. The fungal mitochondrial genome project: evolution of fungal mitochondrial genomes and their gene expression. Curr. Genet. 31, 380–395 (1997).

    CAS  Article  Google Scholar 

  8. 8

    Berbee, M. L. & Taylor, J. W. Dating the evolutionary radiations of the true fungi. Can. J. Bot. 71, 1114–1127 (1993).

    Article  Google Scholar 

  9. 9

    Keogh, R. S., Seoighe, C. & Wolfe, K. H. Evolution of gene order and chromosome number in Saccharomyces, Kluyveromyces and related fungi. Yeast 14, 443–457 (1998).

    CAS  Article  Google Scholar 

  10. 10

    Forsburg, S. L. The best yeast. Trends Genet. 15, 340–344 (1999).Summarizes some differences in the biology of the two yeast species.

    CAS  Article  Google Scholar 

  11. 11

    Guthrie, C. & Fink, G. R. (eds) Guide to yeast genetics and molecular biology. Methods Enzymol. 194, 1–863 (1991).Describes more specific methods and protocols for both yeast species.

    Google Scholar 

  12. 12

    Moreno, S., Klar, A. & Nurse, P. Molecular genetic analysis of the fission yeast Schizosaccharomyces pombe. Methods Enzymol. 194, 795–823 (1991).

    CAS  Article  Google Scholar 

  13. 13

    Kumar, A. & Snyder, M. Emerging technologies in yeast genomics. Nature Rev. Genet. 2, 302–312 (2001).The genomics revolution complements the classical genetics approach.

    CAS  Article  Google Scholar 

  14. 14

    Hoffman, C. S. & Welton, R. Mutagenesis and gene cloning in Schizosaccharomyces pombe using nonhomologous plasmid integration and rescue. Biotechniques 28, 532–539 (2000).

    CAS  Article  Google Scholar 

  15. 15

    Chua, G., Taricani, L., Strangle, W. & Young, P. G. Insertional mutagenesis based on illegitimate recombination in Schizosaccharomyces pombe. Nucleic Acids Res. 28, E53 (2000).

    CAS  Article  Google Scholar 

  16. 16

    Grallert, B., Nurse, P. & Patterson, T. E. A study of integrative transformation in Schizosaccharomyces pombe. Mol. Gen. Genet. 238, 26–32 (1993).

    CAS  PubMed  Google Scholar 

  17. 17

    Hughes, T. R. et al. Widespread aneuploidy revealed by DNA microarray expression profiling. Nature Genet. 25, 333–337 (2000).

    CAS  Article  Google Scholar 

  18. 18

    Wolfe, K. H. & Shields, D. C. Molecular evidence for an ancient duplication of the entire yeast genome. Nature 387, 708–713 (1997).

    CAS  Article  Google Scholar 

  19. 19

    Giaever, G. et al. Genomic profiling of drug sensitivities via induced haploinsufficiency. Nature Genet. 21, 278–283 (1999).

    CAS  Article  Google Scholar 

  20. 20

    Hartwell, L., Culotti, J. & Reid, B. Genetic control of the cell division cycle in yeast. I. Detection of mutants. Proc. Natl Acad. Sci. USA 66, 352–359 (1970).

    CAS  Article  Google Scholar 

  21. 21

    Nurse, P. Genetic control of cell size at cell division in yeast. Nature 256, 547–551 (1975).

    CAS  Article  Google Scholar 

  22. 22

    Nurse, P. Universal control mechanism regulating onset of M phase. Nature 344, 503–508 (1990).

    CAS  Article  Google Scholar 

  23. 23

    Enoch, T., Carr, A. M. & Nurse, P. Fission yeast genes involved in coupling mitosis to completion of DNA replication. Genes Dev. 6, 2035–2046 (1992).

    CAS  Article  Google Scholar 

  24. 24

    Li, R. & Murray, A. W. Feedback control of mitosis in budding yeast. Cell 66, 519–532 (1991).

    CAS  Article  Google Scholar 

  25. 25

    Hoyt, M. A., Totis, L. & Roberts, B. T. S. cerevisiae genes required for cell cycle arrest in response to loss of microtubule function. Cell 66, 507–517 (1991).

    CAS  Article  Google Scholar 

  26. 26

    Koshland, D., Kent, J. C. & Hartwell, L. H. Genetic analysis of the mitotic transmission of minichromosomes. Cell 40, 393–403 (1985).

    CAS  Article  Google Scholar 

  27. 27

    Hieter, P., Mann, C., Snyder, M. & Davis, R. W. Mitotic stability of yeast chromosomes: a colony color assay that measures nondisjunction and chromosome loss. Cell 40, 381–382 (1985).

    CAS  Article  Google Scholar 

  28. 28

    Rine, J. Gene overexpression in studies of Saccharomyces cerevisiae. Methods Enzymol. 194, 239–251 (1991).

    CAS  Article  Google Scholar 

  29. 29

    Stevenson, L. F., Kennedy, B. K. & Harlow, E. A large-scale overexpression screen in Saccharomyces cerevisiae identifies previously uncharacterized cell cycle genes. Proc. Natl Acad. Sci. USA 98, 3946–3951 (2001).

    CAS  Article  Google Scholar 

  30. 30

    Herskowitz, I. Functional inactivation of genes by dominant negative mutations. Nature 329, 219–222 (1987).

    CAS  Article  Google Scholar 

  31. 31

    Prelich, G. Suppression mechanisms: themes from variations. Trends Genet. 15, 261–266 (1999).

    CAS  Article  Google Scholar 

  32. 32

    Guarente, L. Synthetic enhancement in gene interaction: a genetic tool come of age. Trends Genet. 9, 362–366 (1993).References 28 and 30 are classic reviews describing the main features of the screens covered in this article and providing some theoretical background.

    CAS  Article  Google Scholar 

  33. 33

    Booher, R. & Beach, D. Interaction between cdc13+ and cdc2+ in the control of mitosis in fission yeast: dissociation of the G1 and G2 roles of the cdc2+ protein kinase. EMBO J. 6, 3441–3447 (1987).

    CAS  Article  Google Scholar 

  34. 34

    Sandrock, T. M., O'Dell, J. L. & Adams, A. E. Allele-specific suppression by formation of new protein–protein interactions in yeast. Genetics 147, 1635–1642 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35

    Tye, B. K. & Sawyer, S. The hexameric eukaryotic MCM helicase: building symmetry from nonidentical parts. J. Biol. Chem. 275, 34833–34836 (2000).

    CAS  Article  Google Scholar 

  36. 36

    Hardy, C. F. J., Dryga, O., Seematter, S., Pahl, P. M. B. & Sclafani, R. A. mcm5/cdc46-bob1 bypasses the requirement for the S phase activator Cdc7p. Proc. Natl Acad. Sci. USA 94, 3151–3155 (1997).

    CAS  Article  Google Scholar 

  37. 37

    Lee, M. G. & Nurse, P. Complementation used to clone a human homologue of the fission yeast cell cycle control gene cdc2. Nature 327, 31–35 (1987).

    CAS  Article  Google Scholar 

  38. 38

    Grallert, B. & Nurse, P. An approach to identify functional homologues and suppressors of genes in fission yeast. Curr. Genet. 32, 27–31 (1997).

    CAS  Article  Google Scholar 

  39. 39

    Wittenberg, C., Sugimoto, K. & Reed, S. I. G1-specific cyclins of S. cerevisiae: cell cycle periodicity, regulation by mating pheromone and association with the p34CDC28 protein kinase. Cell 62, 225–237 (1990).

    CAS  Article  Google Scholar 

  40. 40

    Hennessy, K. M., Lee, A., Chen, E. & Botstein, D. A group of interacting yeast DNA replication genes. Genes Dev. 5, 958–969 (1991).

    CAS  Article  Google Scholar 

  41. 41

    Kroll, E. S., Hyland, K. M., Hieter, P. & Li, J. J. Establishing genetic interactions by a synthetic dosage lethality phenotype. Genetics 143, 95–102 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  42. 42

    Pasion, S. G. & Forsburg, S. L. Nuclear localization of fission yeast Mcm2/Cdc19p requires MCM complex formation. Mol. Biol. Cell 10, 4043–4057 (1999).

    CAS  Article  Google Scholar 

  43. 43

    Clarke, L. Centromeres: proteins, protein complexes, and repeated domains at centromeres of simple eukaryotes. Curr. Opin. Genet. Dev. 8, 212–218 (1998).

    CAS  Article  Google Scholar 

  44. 44

    Sikorski, R. S. & Boeke, J. D. In vitro mutagenesis and plasmid shuffling: from cloned gene to mutant yeast. Methods Enzymol. 194, 302–318 (1991).

    CAS  Article  Google Scholar 

  45. 45

    Liang, D. T. & Forsburg, S. L. Characterization of S. pombe mcm7+ and cdc23+ (MCM10) and interactions with replication checkpoints. Genetics (in the press).

  46. 46

    Johnston, M. The yeast genome: on the road to the Golden Age. Curr. Opin. Genet. Dev. 10, 617–623 (2000).

    CAS  Article  Google Scholar 

  47. 47

    Mullen, J. R., Kaliaraman, V., Ibrahim, S. S. & Brill, S. J. Requirement for three novel protein complexes in the absence of the Sgs1 DNA helicase in Saccharomyces cerevisiae. Genetics 157, 103–118 (2001).An excellent example of a synthetic lethal screen and plasmid shuffle analysis.

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

I thank L. Pillus for helpful comments on the manuscript. I am a scholar of the Leukemia Society of America. Support in my lab comes from the National Institutes of Health, the National Science Foundation and the American Cancer Society.

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DATABASE LINKS

actin

Cdc7

MCM5

Cln1

Cln2

Cln3

ADE2

ade6 +

LEU2

CAN1

URA3

TRP1

SGS1

ade3

slx1–6

cdc28

YEAST PHYLOGENY

Ascomycota

Fungal phylogeny

GENOME DATABASES

Saccharomyces Genome Database (SGD)

Sanger Centre S. pombe database

Proteome (YPD and pombePD)

PROTOCOLS

S. cerevisiae

S. pombe

OTHER YEAST RESOURCES

Yeast virtual library

Gene conversion table

Saccharomyces genome deletion project

TRIPLES

LABS AND INVESTIGATORS

S. cerevisiae labs

S. pombe labs

Susan Forsburg's lab

COMMUNITY INFORMATION

S. cerevisiae

S. pombe

Glossary

ASCOMYCETE

Free-living fungus that reproduces sexually through the formation of spores packaged in a sac called an ascus. Some taxonomists include non-sexually reproducing fungi with DNA sequences, which indicates a close degree of relatedness.

COMPLEMENTATION TEST

Determines whether two recessive mutations are in the same functional unit or gene. Two recessive mutant strains, a1 and a2, crossed together complement each other if the resulting diploid has a wild-type phenotype; as each provides the function missing in the other, they are assumed to affect independent genes. If, instead, the diploid has the mutant phenotype, then a1 and a2 do not complement and are assumed to affect the same gene.

RECOMBINATION

Any process in a diploid or partially diploid cell that generates new gene or chromosomal combinations not found in that cell or in its progenitors. At meiosis, recombination (or crossing over) is the process of reciprocal exchange between homologous chromosomal segments that generates a haploid product genotypically distinct from the two haploid genotypes of the original meiotic diploid.

EPISTASIS

The phenotype caused by a mutation in one gene is masked by a mutation in another gene. Epistatic analysis requires that two mutants have distinguishable phenotypes. It can be used to determine the order of gene function by testing whether the phenotype of the double mutant ab is similar to that of mutant a, or mutant b.

HETEROLOGOUS RECOMBINATION

Recombination between DNA molecules with significantly different sequences, for example when a transgenic construct integrates randomly in the genome.

HAPLOINSUFFICIENCY

A gene dosage effect that occurs when a diploid requires both functional copies of a gene for a wild-type phenotype. An organism that is heterozygous for a haploinsufficient locus does not have a wild-type phenotype.

REPLICA PLATING

A classic method to duplicate the colonies on an agar plate by stamping them on sterile velvets or filters, and then applying these copies to new (replica) plates. The replica plates can then be used to test the colonies for growth on different nutrient media or at different temperatures.

HIGH-COPY LIBRARY

Most plasmid episomes in yeast are present at greater than one copy per cell, leading to an increased dosage of any gene(s) carried by the plasmid. The high copy dosage effect can be enhanced if the cells are transformed with a library that contains cDNAs expressed by strong promoters.

EPISOME

An independent DNA element, such as a plasmid, that can replicate extrachromosomally or that can be maintained by integrating into the genome of the host.

DOMINANT NEGATIVE

A mutant allele that interferes with the function of its wild-type version.

SYNTHETIC INTERACTIONS

These occur when a double mutant has a phenotype different from either single mutant parent. For suppressors (synthetic viable), the double mutant is viable when at least one of the single mutants is not. For synthetic lethal mutants, the double mutant is inviable under conditions in which both parents are viable.

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Forsburg, S. The art and design of genetic screens: yeast. Nat Rev Genet 2, 659–668 (2001). https://doi.org/10.1038/35088500

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