Essential gene analysis in quire: An introduction

All organisms, including humans, have many genes that are essential for growth. The budding yeast Saccharomyces cerevisiae has around 6,000 genes and about 1,000 of them are essential. Essential genes in S. cerevisiae are involved in many core biological functions, such as DNA replication, transcription, translation, protein degradation, and maintenance of organelles.

Identifying essential S. cerevisiae genes

The gold standard for identifying which genes are essential involve making a heterozygous deletion in a diploid strain, inducing sporulation, and performing tetrad dissection so that the fate of each meiotic product can be followed. A 2:2 pattern of growth:inviability in the tetrad demonstrates that the deleted gene is essential.

Random spore analysis of the sporulated heterozygous diploids is more problematic than tetrad dissection. If random spore analysis is used, spores should be plated such that the total number of viable spores can be counted in addition to those containing a deletion marker. If the gene is non-essential, a roughly 50:50 mix of haploid spore clones with and without the deletion should be observed. If the gene is essential, only haploid spore clones without the deletion should grown. However, even if the gene is essential, colonies can be observed on selective plates. These result from those clones that have retained a copy of the essential gene. For example, diploids that inappropriately survive the isolation procedure will grow (although these can be identified in a variety of ways). More problematically, strains that are disomic for the chromosome containing the the deletion of the essential gene due to meiotic nondisjunction can be selected for. PCR amplification of an internal region of the deleted gene is a rapid way to identify clones retaining the deleted gene. Thus, if the fraction of colonies containing the deletion marker is far less than that predicted from the expected 50:50 ratio, then it is likely that the gene is essential. Verification should probably involve tetrad dissection.

Working with essential genes

Most yeast genetics involves the analysis of mutations. For essential genes, the most common mutations are "temperature sensitive", e.g. they retain enough of the function at a "permissive" temperature, but become inactivated when the temperature is raised to a "non-permissive" temperature. Temperature sensitive mutations are often not entire normal even at permissive temperatures. Another strategy for working with essential genes is to down regulate their expression; a systematic approach has been through the DAmp allele collection, which integrates a marker into the 3' end of the UTR to destabilize the mRNAs. Experience with the DAmp allele collection has shown, however, that not all DAmp alleles are equally suppressed.
The definition of essential genes is context dependent

Whether or not a gene is required for growth can depend on many factors including the nutrients available in the growth medium. For example, the LYS2 gene is required for growth on media lacking lysine. Because of genes like this, essential genes are defined as being required for growth on rich media. Thus, LYS2 is not an essential gene, but rather causes a lysine auxotrophy.

But even for genes that are required for growth on rich media, the genetic context matters. Deletion of three genes in the DNA damage sensing pathway, MEC1, DDC2 (also called LCD1), and RAD53 are all inviable. The essential function of these genes, however, is in a phosphorylation cascade that phosphorylates the Sml1 ribonucleotide reductase inhibitor, which leads to its degradation. Thus the inviability of deletions of MEC1, DDC2, and RAD53 can all be suppressed by simultaneous deletion of SML1. Similarly, deletion of a gene involved in Okazaki fragment maturation, DNA2, can be suppressed by deletion of the gene encoding the Pif1 DNA helicase.

Conversely, some non-essential genes can be made to be essential in genetic contexts when other genes are inactivated. For example, deletion of SLX1 alone or deletion of SGS1 alone results in viable cells. However, simultaneous deletion of both SLX1 and SGS1 is inviable. This is termed "synthetic lethality" (e.g. it is a lethality that is created "synthetically" by combining mutations), and is useful for understanding the roles of genes. Synthetic genetic interactions giving rise to growth defects on rich media have now been pursued for almost all S. cerevisiae genes. In addition, synthetic lethality currently being explored for use in targeting cancers. The "simple" model for synthetic lethality is that both genes are redundant for some essential process; however, the vast majority of identified interactions do not fit this model.

Because of these complexities, the list of essential genes in S. cerevisiae is somewhat dependent on the strain background (RTT105 is essential in the W303 background, but not in the S288c background). A remarkable illustration of this has been observed by the systematic shuffling of a synthetically reconstructed S. cerevisiae genome (Sc2.0), in which the pattern of essential genes depends on which genes have been lost and retained during the genomic shuffling. Similar complexity has been observed in attempting to define a "core" group of essential human genes using primarily cancer cell lines.

Next steps

The next blogs will use of the quire programming language to explore different aspects of the essential S. cerevisiae genes. This will illustrate both the use of quire to perform genetic analyses and aspects of the S. cerevisiae essential genes.