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The art and design of genetic screens: yeast

Nature Reviews Genetics volume 2, pages 659–668 (2001)Cite this article

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.

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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.

Author information

Authors and Affiliations

  1. Molecular and Cell Biology Laboratory, The Salk Institute, 10010 N. Torrey Pines Road, La Jolla, 92037, California, USA

    Susan L. Forsburg

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  1. Susan L. Forsburg
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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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