Vastly used in molecular biological research, a the yeast species Saccharomyces cerevisae is one of the most studied unicellular organisms. Its structure is complex and close to one of animals and plants, despite their relatively small size of 3-5 µm. They can be grown on agar plates or in liquid cultures. The genome of Saccharomyces cerevisae consists of 16 chromosomes, 1.4·107 bp in total.
S. cerevisae cells usually have an almost round form. With a generation span of 1.5 hours they reproduce through an asymmetric division process called budding. The yeast cells can exist in both haploid state (one copy of genome) and diploid state (two copies of genome) with the diploid state being the preferred one . In both states the cells can reproduce by mitosis. There exist 2 mating types of S. cerevisae – MATa and MATα with each type producing a pheromone that binds to a specific receptor in the opposite mating type. When the two mating types are placed near, after recognition of the signal pheromone by the receptor, a cascade is set in progress, which results in merging into one diploid cell. With nutrients present the diploid cell can divide through mitosis. In case of starvation or other stress the diploid cells undergo meiosis and produce 4 haploid spores.Cell cycle of SaccaromycesSaccharomyces cerevisaecerevisiae . The two mating types MATa and MATα can reproduce through budding, mitotically. When cells of both types are present, a process call conjugation is started and the cells merge into a diploid cell that can also reproduce mitotically by budding. In case of stress, such as carbon and nitrogen starvation, the diploid cells can undergo meiosis and thus produce four haploid spores. When conditions improve, the spores can start reproducing as haploids and then merge into diploids again.
So called ade-mutations are mutations in enzymes that play an important role in purine biosynthesis in yeast. These mutations make the cells unable to synthesize purine bases adenine and guanine and thus are auxotroph toward.
In the yeast cells with mutations in ade1 or ade2 a compound called 5-aminoimidalzolribotide or AIR is accumulated. Through polymerization of AIR a red pigment is created. So called cdc mutants have a mutation in genes that are directly or indirectly involved in cell division cycle . These mutants are temperature sensitive, which means that at room temperature these genes are active and are inactivated at 37°C. The cells are then fixed at a certain point of the cell cycle .
Materials and methods
The following ingredients were mixed in 500 ml water to create YPD medium: 5 g yeast extract, 10 g bacto peptone, 10 g glucose, 10 g bacto agar and 40 mg adenine. The beaker was autoclaved for 10 minutes; the solution was then tempered at 45°C. The warm solutions were poured into petri dishes under sterile conditions.
The six yeast strains were put onto YPD petri dishes and then put to incubation at 30°C. Six plates from other groups were then taken and put onto one SC- and one SC+ plate with an inoculation loop, creating a complementation pattern as illustrated in Figure 3. In the cross sections the two probes were mixed with the inoculation loop. The plates were then incubated at 30°C for several days. For the analysis the plates of other groups were taken.
For the analysis plates of other groups were used. With a sterile pick a colony was taken and in 150 μl water suspende d. Then 10 μl thereof were taken and put onto a coverslip. With a light microscope and a picture of the colony was then taken.
The results of the complementation scheme. On the left is the CD- plate, whose medium didn’t contain adenine, on the right is the CD+ plate, whose medium did contain adenine. Six strains (Tab. 1), of them 3 MATa and 3MATα were crossed. The yeast strains were put according to the scheme in Figure 3. For each strain a reference point was put, so that a haploid colony would grow, to see if the diploid form changed its phenotype. In every cross section the two haploid strains were mixed with an inoculation loop.
On the CD+ plate all the colonies, both haploid and diploid, were white. On the CD- plate red colour could only be observed in case of haploid α ade2 strain, and traces of red in case of its mixing with a-mating type strains ade2 and ade1/ade6. Both ade1 strains had a very light pink colour. The same colour could be observed in a+α ade1 mix, patches of it were observed in case of α-ade1 with a-ade2, α-ade1 with a-ade1/ade6 and α-ade6 with a-ade1 mixes. All other mixes were completely white or white with patches mentioned above.
A colony of cdc28-1, incubated at 30°C. It serves as a model colony at 30°C, because the mutations aren’t visible as all proteins are expressed. Different phases of mitosis can be observed, some examples are marked. Cell 1 is not dividing, cell 2 has started dividing and as in the process of budding, a little bud can be seen. Cell 3 has almost finished dividing: the mother and daughter cell are almost separated.
Complementation occurs when two haploid strains have the same phenotype, but caused by mutations in different genes. By merging together, a diploid is created. The genes coding enzymes that are not expressed are defect on one chromosome set, but are intact on the other and thus both needed enzymes are produced. The diploid cell has then the phenotype of an intact and not mutated cell. In case of SC+ plate all the strains are white. The explanation would be that the cells didn’t use the de novo purine biosynthesis pathway. There are salvage pathways, through which adenine is transferred onto phosphoribosyl pyrophosphate by adenine-phosphoribosyl pyrophosphate transferase to produce AMP. ATP can be then produced and through degradation pathway transformed into inosine triphosphate and then guanine triphosphate. The needed quantities of purines are thus provided, diminishing the need to use de novo pathways by which red pigments could be produced. The cells therefore remain white. The experiment showed the predicted result, all colonies and diploid mixes where white.
Ade1 and ade 2 strains have a mutation that cause malfunction in SAICAR synthetase and AIR carboxylase respectively, because of which AIR is accumulated and then polymerised into a red pigment. Ade 6 causes a mutation in FGAM synthetase, a protein catalysing a reaction up the pathway and thus no AIR is produced, cells remain white. In case of ade1/ade1 and ade2/ade2 diploid cells neither of chromosomes codes a functioning protein, thus the red phenotype is preserved. In case of ade1/ade6 strain, mixing it with ade 1 makes the diploid cell able to produce FGAM synthetase and thus AIR but then it accumulates as none of chromosomes codes for a functioning AIR carboxylase (ade1 gene).
In reality while the CD+ plate gave the expected results the CD- plate looked different from what was expected. The only haploid red strain was α ade2, a ade2 was white, ade1 strains were slightly pink. The diploids containing α ade6 looked as expected, the diploids containing α ade2 also looked as expected, but the corners in case of mix with a ade1/6 were red, which could mean that a part of cells didn’t undergo conjugation to diploid state. The same could be said about α ade1 with a ade 2 mix. Ade 1 mixes had the same pinkish colour. The a ade2 haploid strain was completely white. A possible reason could be that the cells somehow mutated again and thus obtained the needed protein to function. This could also explain why ade2 / ade2 mix was white (apart from the red crust from haploid α strain), as the diploid cells could use the a ade2 genome to function properly. The white colour could also mean that a different and thus wrong strain was put onto a petri dish to cultivate, from which it was transferred onto the pattern.
The cell forms of cdc mutants coincide with those acquired by Hartwell et al. In case of cdc28-1 the replication of DNA is impaired. This is also the case of cdc4, where cells produce unusually large bud, which was observed in the microscope . Cdc19 mutants arrest cell cycle at G1 interphase of division . The cdc9 mutants stop at medium stage of nuclear division. In all strains cells with black dots inside were observed, that could be cells in process of apoptosis.