The human genome is often compared to a cell instruction manual – if each page were to represent one of the estimated 25,000 distinct genes, it would claim the title of the longest book ever to be written. These gene units are coded for by a unique fingerprint sequence of up to several million bases, each referred to by one of four basic letters: A, T, C or G. Despite this manuscript of life existing within most somatic cells, it was only in 2003 that the completion of the thirteen year-long Human Genome Project was able to cast light upon a genetic map that would later prove essential in navigating scientific research through the labyrinth of the genome. While the success of this project is widely lauded, the pivotal and unexpected role of S. cerevisiae (yeast) often remains in the shadows.
The Importance of Yeast
Despite the title of the Human Genome Project implying that research focused on human cells, yeast rapidly rose to the centre of mapping techniques for its unique properties that distinguished it from other candidates more similar to humans. While human genetic pedigrees – genetic trees used to display the Mendelian patterns of trait heritance - may be useful in analysis, the crevasse of time between generations stunted its potential applications and called for a faster-reproducing organism. The budding time of yeast averages 90 minutes, meaning that the trends in genetic composition and the occurrence of de novo (new) mutations from generation to generation could easily be observed. Furthermore, yeast has the potential to exist in both a diploid and a haploid form in relation to the environmental conditions; this permits researchers to initiate either sexual or asexual reproduction in a certain colony to monitor differences between these modes of replication.
Tetrad Formation
Yeast genes are mapped while it is in haploid – halved genetic material - form, requiring the yeast to sporulate under nitrogen-deficient conditions and create a tetrad of meiotically-divided haploids. This is performed via the following general method:
The yeast sample is first streaked upon a petri dish and incubated, allowing for budding. Each colony appears as a distinct patch of yeast growth; a single colony is then isolated and swirled in a minimal media consisting of salts, minerals, a sugar source, and the absence of nitrogen. Under this lack of nitrogen, the yeast colony undergoes the evolutionary process of sporulation in response to stressful conditions to form an ascospore which would – in the wild – be able to drift to a more nitrogen-rich location. This is achieved as the cells exit the mitotic cell cycle of normal cell division and initiate meiosis within the nuclear envelope. During meiosis, the genetic material divides twice in succession, resulting in four daughter cells, known collectively as a tetrad. The membrane of the mother cell persists around the tetrad, acting as a protective ascus coating around the four inner spores.
To reach the haploids for study, enzymes are employed for the dissolution of this ascus. The cells may then be observed using a powerful tetrad-dissecting microscope equipped with a fine glass needle designed to isolate the individual haploids from the tetrad.
Using Tetrads to Measure Gene Linkage
During the meiotic process, the genes do not segregate into identical cells as they would during mitosis. Instead, genetic variety of offspring is caused by recombination: this is the crossing over of DNA between different chromosomes to exchange genetic material at a certain point. Two genes are usually selected for observation to determine their genetic distance, and thus position within the yeast genome. The closer together the genes are, the more likely they are to remain on the same chromosome and in the same daughter cell following recombination.
Without recombination, all haploids have what is known as the ‘parental ditype’ genotype; this is identical to that of the mother cell. A potential genotype of the mother cell could be AB/ab, in which A and a are two alleles of the same gene (as are B and b) and AB and ab represent the combinations of these alleles present on each chromosome belonging to a pair. If recombination does not occur between the loci of the two genes on the chromosomes, all haploid daughter cells have either an AB or ab genotype, which matches that of the mother chromosomes.
However, in the event of recombination, one of two different offspring types may arise. The first is the non-parental ditype, in which none of the daughter cells have chromosomes that match the mother cell, as recombination has switched the arrangement of the two genes. This would be represented by a mix of Ab or aB haploids. The second is the tetratype, with four different possible genotypes – two of which are recombinant and two are parental. Therefore, the daughter cells would exhibit a mix of AB, ab, Ab and aB genotypes. Both these types of tetrads show that the chromosomes have crossed over and swapped material at some point between the genes A and B.
Observing these haploids is critical in the measurement of genetic distance between yeast genes, since the relative numbers of each type of tetrad (parental ditype, non-parental ditype and tetratype) can be directly input into this formula, from which genetic distance measured in centimorgans (cM) can be derived:
Genetic Distance = 100 x (T + 6NPD)/(2E)
where T corresponds to the number of tetratypes, NPD to the number of non-parental ditypes and E to the total number of haploid cells in the sample. As recombination events increase in frequency, the numerator of the fraction rises since the number of tetratypes and non-parental ditypes increases in relation to the total number of cells. This causes the overall fraction to increase, displaying a proportional increase in genetic distance.
Applications
Although yeast seems an unlikely subject to map genetic distances and determine the degree to which genes are linked, it is to this unique organism that the Human Genome Project owes its success. While morphologically, humans and yeast are highly distinguishable, 23% of genes are homologous between these two species and observations of genetic distances in yeast are frequently mirrored in the human genome. These genetic distances can – like distances on a geographical map – be used to physically place the genes relative to each other to construct a highly accurate sequencing of bases.
The most notable application of yeast technology resides in the study of genetic markers: these more visible and easily identifiable ‘flags’ are linked to and signal the presence of other, more significant alleles and mutations close by on the same chromosome. Among the marker loci identified using yeast are even genes which point towards antibiotic resistance in bacteria – a corner of research with the future potential to revolutionise healthcare and accelerate pharmaceutical evolution.
References
MITx 7.03.1 Genetics: The Fundamentals
accessed: 19th November 2023
https://www.uvm.edu/~dstratto/bcor101/mapping3.htm
Accessed: 27th December 2023
K-State Parasitology Laboratory: Mendelian Genetics Problems
Accessed:
https://www.k-state.edu/parasitology/biology198/answers2.html 27th December 2023
A. Neiman: Ascospore Formation in the Yeast Saccharomyces cerevisiae
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https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1306807/#:~:text=The%20presence%20of%20a%20poor,%2C%20and%20sporulate%20(40). 27th December 2023