Saccharomyces cerevisiae (hereafter simply referred to as “yeast”) is one of the most intensively studied eukaryotic organisms. In fact, it is probably one of the oldest domesticated ones, being used for beer brewing already in Sumeria and Babylonia around 6000BC and in old Egypt for dough leavening.
In the 1930s, it was recognized as an ideal model system in which to study several aspects of eukaryotic cell biology, competing with the by then very popular Neurospora crassa, (a filamentous fungus which we have discussed before due to its importance in the history of molecular biology, see here), and is now at the forefront of experimental molecular biology.
Many of the widely used methods in the field have been pioneered in yeast (gene expression microarrays, for example) and it continues to be the major proving ground for large-scale studies and a wellspring of new discoveries.
Yeast, a single-celled hemiascomycetes fungus, combines several advantages that makes it particularly suitable for cell and molecular biology studies, including rapid growth, short generation time, tractable genetics, well-known physiology, a variety of established vector and transformation systems, high-efficiency homologous recombination (which in itself could be used as a tool for the study of gene function in other organisms, see here) and a collection of KO mutants (see below), just to name a few. Notably, yeast was the first eukaryotic organism to have its genome sequenced1 and this information, along with all the experimental advantages that yeast possesses, have proven instrumental for the advancement of the study of gene function not only in yeast, but in other organisms as well, including humans.
As mentioned, a lot of what we now know about yeast comes from high-throughput studies that have been pioneered in yeast, and notably, many of them have been later on applied to other systems.
The study of gene function, a logical step after genome sequencing, encompasses a combination of several complementary approaches, including subcellular localization, protein-protein interactions, genetic interactions, gene expression profiles and KO phenotyping (just to name a few), and all of them can be (and have been) scaled up to study gene function globally in this organism.
In this two-post series on yeast, I will discuss a very clever methodology developed for the high-throughput study of KO strains in Saccharomyces cerevisiae. In this first post, I will talk about how KO strains are generated and in the second one, how they are studied globally. Through these methodologies, it’s possible to study which genes are important for growth under a variety of different conditions in this unicellular organism, which has helped us understand the role of many genes in yeast that we knew nothing about. The information derived from these studies has also helped us understand gene function in other organisms, including humans, and has had wide implications, for example, for the study of drug action.
The global approach I will discuss, pioneered by the laboratory of Ron Davis2, is based on tagging each KO strain and then using that tag to evaluate how the KO strains are doing under different growth conditions over time. This brief explanation will make a lot more sense once I describe how the KO strains are generated and how the importance of each gene for growth under different conditions, is scored.
In the interest of brevity, I won’t do a historical perspective of the methodology, but just describe the way the mutants were generated as part of the Yeast Deletion project and how they have been used.
A general scheme of the methodology for KO generation is shown on figure 1, which represents a “PCR-based gene deletion strategy”.
The idea of the Yeast Deletion Project is to generate a start- to stop- codon deletion of each of the ORFs in the yeast genome, by replacing it with a kanMX module (which confers resistance to G418).
In order to do this, you’ll need to target the kanMX module specifically to each locus. How is this specificity attained?
This is when two rounds of PCR kick in.
In the first round, you will attach something to the common kanMX module. The primers for this are long (74mers) and are divided into functional regions.
Primers are specifically designed to flank the start and stop codons of each ORF in the genome. The forward primer contains 18 bases of homology to the region upstream of the target ORF and the reverse one, 18 bases of homology to the region downstream of the target ORF (these are depicted as the “ATG” and “TAA” regions in the primers in figure 1). Appended to these sequences, there are unique 20-base sequence tags (labelled UPTAG and DNTAG in figure 1) and common priming sites (U1-U2 in the forward primer and D1-D2 in the reverse one). The importance of these sequences will be discussed below.
Note that the common tag priming sites U2 and D2 are homologous to the 5′ and 3’ regions of the kanMX4 module, respectively. This allows for the primers to anneal to the cassette for Round 1 of PCR. The importance of the U1 and D1 sequences will be discussed in the next post of the series.
After that round of PCR, you are left with a kanMX cassette that is flanked by specific tags and by sequences that flank a particular ORF in the genome.
A second round of PCR comes next, aimed at extending the cassette’s homology region to the yeast genome. This greatly increases the specificity in the homologous recombination step (see figure 1 and below).
By the end of the second round of PCR, you are left with a kanMX cassette flanked by specific tags and by sequences that will allow its specific integration in the genome.
Now, we are ready to transform yeast. The cassette is transformed into diploid yeast cells where, by homologous recombination, will replace one copy of the WT gene (the possibility of replacing both alleles in the same cell in one transformation event is extremely low).
Transformation is done into diploid strains in case the gene being knocked out is essential. Growth in media containing G418 is used to select for transformants and correct integration is later checked by PCR.
The resulting transformants are heterozygous diploid deletion strains (harboring one WT allele and one disrupted one). How do we get the homozygous deletion strains?
Simple. We just sporulate the diploids (induce diploids to go through meiosis).
The KO procedure described, when applied to a non-essential gene, produces four viable haploid spores: two contain the intact ORF and two contain the cassette.
Homozygous diploid strains, which are also available, are constructed from the mating of two independently isolated haploid mutants.
What about essential genes? Applying the KO procedure to these genes and inducing sporulation, yields only two viable spores, each containing the wild-type ORF (i.e. yeast haploid cells with the disrupted allele will die). In any case, this tells us something about that gene that we didn’t know: it is essential under those growth conditions. The methodology for essential genes then, only proceeds up to the heterozygous diploid stage
Note that “non-essential does not mean “without a function” or “non-important”: deletion of “non-essential” genes can also have a negative impact on fitness (resulting for example, from the imbalance of gene product), but under the conditions of selection used in the project, their deletion does not lead to lethality (although they could be sick) and homozygous deletion strains for these genes can be obtained.
The Yeast Knockout collection thus contains deletion strains in four different backgrounds: haploids of each mating type (there are two of them, “a” and “alpha”), homozygous diploids (for non-essential genes) and heterozygous diploids.
In this way, more than 90% of the ORFs larger than 100 amino acids as well as verified shorter ORFs have been disrupted, providing the research community with an invaluable tool for studying gene function.
As I mentioned before, yeast is particularly amenable to high throughput studies and many of the global approaches used in other organisms have been pioneered in yeast. How can we then study gene function globally in this organism? How can we evaluate which genes are important under different growth conditions on a high-throughput manner? One approach, which we will discuss in the next post of this series, makes use of the Yeast Deletion Collection, particularly, of the unique tag each strain contains.
1 Goffeau A, et al. (1996) Life with 6000 genes. Science 274 (5287): 546-567.
2 Shoemaker DD et al. (1996) Quantitative phenotypic analysis of yeast deletion mutants using a highly parallel molecular bar-coding strategy. Nat Genet. 14(4):450-6.
3 Chu AM, & Davis RW (2008). High-throughput creation of a whole-genome collection of yeast knockout strains. Methods in molecular biology (Clifton, N.J.), 416, 205-20 PMID: 18392970