Kathleen Swanson BIO210L Lab Summary: Yeast October 27, 2013

Introduction:

DNA is the fundamental building blocks of all life. These molecules are made of nucleic acid bases (adenine, guanine, tyrosine, and cyanine). The Central Dogma: DNA is translated to RNA which then encodes for specific proteins, is fundamental to all studies of life and molecular genetics. DNA is a transferable, inheritable material as confirmed by the Avery Experiment. These experiments used bacterial transformation in mice to prove that virulent DNA molecules were being transformed, or transferred into benign bacteria that did not contain any virulent material. After this transfer of genetic material the once benign bacteria was capable of causing disease and lethal to the mice used for the protocol. Understanding the transfer of genetic information is crucial to continued advancements in pharmacology and medicine, disease control and prevention, and genetic counseling and diagnosis/confirmation of genetic disorders that allow for medical professionals to maximize their treatment plans for these conditions.

This experiment utilizes the eukaryotic cell yeast (a fungus) as the model organism of choice. In addition to its usefulness in baking and fermentation of alcohol, the use of yeast cells in laboratories has many advantages Yeast cells are non-pathogenic, grow at a rapid rate, can exist in haploid or diploid states, and have the ability to receive or drop plasmids (tiny circular DNA that are independent of the chromosomal DNA of yeast and bacterial cells). Yeast can also thrive, grow and reproduce in many environments in part due to their ability to produce adenosine triphosphate (ATP) from carbon and sugars, both aerobically (with oxygen) and anaerobically (without oxygen). The ability of yeast cells (or any cell) to acquire plasmid DNA is a feature that increases the sustainability of the species even under less habitable conditions. Transformation of plasmid DNA, a horizontal transfer of genetic material, allows for an immediate remedy needed to survive hostile environments as opposed to vertical transfers of genetic material to offspring, which requires many generations for the evolutionary changes needed for an organism to adapt to its environment.

Recombination of DNA allows genetic material to be passed from one cell, and permanently integrate into the chromosomal DNA of another. In addition to recombination, yeast cells are also able to pick up

and maintain plasmids that are beneficial to their survival needs. These tiny circular pieces of DNA can be lost or gained at any point as they are free from the chromosomal DNA. This knowledge allows us to ascertain the phenotype of a given yeast cell depending on the choice to either keep a plasmid or lose it once the proteins it encodes, or function it serves, become unnecessary.

The genes ADE2 and ADE3 are both necessary for the production of adenine (one of four nucleic acid bases of DNA molecules) and adesine triphosphate (ATP). This laboratory exercise uses yeast cells that are wildtype for ADE3 are mutated at ADE2. This mutation of ADE2 allows the precursor for adenine and ATP, colored orange, to build up within the cells. Yeast cells that have a mutation at ADE2 will be unable to grow or sustain life due to their inability to make adenine and ATP; but when placed on a media that contains adenine, the mutant cells will continue to grow and survive and will appear orange in color. Wildtype yeast cells for both ADE2 and ADE3 are beige. This distinction in pigments allows us to easily observe the phenotype of both the wildtype and mutant ADE2 cells.

Transformation of plasmids that are wildtype for ADE2 should allow the mutant yeast cells to start producing their own adenine allowing them to live on a media that doesnt contain adenine as well as causing their color to change to beige. Once transformation of the plasmid has occurred in the laboratory, a yeast cell could permanently integrate this ADE2 gene into its chromosomal DNA through recombination, or the intact plasmid may continue to encode for proteins independent of the chromosomal DNA. In the case of the latter, this plasmid can be lost at any point, for as long as it remains separate from the chromosomal DNA. When conditions are such that the yeast cells no longer need to make their own supply of adenine, the cells will lose the plasmid and revert to their mutant state, unable to make adenine and presenting as orange. It is predicted that when the transformed yeast cells are transferred from a normal media to one containing adenine that a large number of the cells will lose their plasmid and revert to their previous mutant (ADE2), orange colored state. Also, it is believed that some of these yeast cells could undergo recombination and permanently integrate the genetic material from the plasmid into their chromosomal DNA, and will remain beige colored as they are no longer mutated at ADE2.

Methods: 500L of yeast cells, mutated at ADE2, were spun down for 5 minutes to allow for separation and removal of the yeast cells from the supernatant (media). The yeast cells extracted were resuspended in 100 L of OneStep Buffer (OSB) which prepares the cell for receiving plasmid DNA by causing small holes in the cell wall. The following were added to the yeast cells suspended in the OSB: 11 L of DTT, 5 L of ssDNA (a nonspecific DNA), and 1 L of plasmid DNA. The addition of DTT and ssDNA (more expendable nonspecific forms of DNA) aid in the transfer of plasmid DNA through the cell wall as transformation is more successful when the yeast cell has a significantly higher concentration of DNA outside the cell. The DNA will move along its concentration gradient ensuring higher yields of the desired plasmid DNA within the yeast cell. The cells were heat shocked

by incubation for 30 minutes at 45C. This increase in temperature also improves the transfer yield of plasmid DNA by creating holes in the cell wall. 100 L of these cells are then transferred to a plate containing normal media (not containing adenine).

In order to determine which yeast cells have undergone recombination as opposed to those who merely took up the plasmid for a period of time, the now visible yeast cells which contain plasmid DNA must be transferred to a new media containing adenine. This transfer will cause the yeast cells that have not undergone recombination to lose their plasmid in the presence of adenine, and make them distinguishable from those that remain beige and continue making their own adenine. After the yeast cells have grown, a 5 L sample of the cells was collected and diluted with 10 mL of H2O. A 50 L sample was transferred taken from this dilution, to the new plate containing adenine (approximately 250 cells per plate). 5 days later orange and beige cells were counted to determine the number of recombination events and plasmid dropped events. Before dilution with water, the concentration of yeast cells was 10 x 106 cells/mL. Dilution with H2O resulted in a new concentration of 5 cells/ L.

Results:

Figure 1: sample of transformed yeast cells (wildtype phenotype for ADE2) on a media that did not contain adenine; Beige colored yeast cells are phenotypically wildtype for ADE2.

Figure 2: beige, orange, and orange-sectored yeast cells, after transformation of wildtype plasmid, growth, and transfer to adenine containing media. Orange cells are phenotypically ADE2 mutants (orange, sectored) and wildtype cells are beige

Figure 1 shows that all yeast cells have grown considerably confirming they are capable of producing their own ATP, and are all beige in color which is only seen in cells that are wildtype for the ADE2 gene. There were no orange cells found (including orange-sectored). Figure 2 shows yeast cells on a media containing adenine. After a growth period of 5 days, 37 total cells were counted: 12 cells were beige colored and 25 cells were orange colored orange-sectored cells. Conclusion:

Yeast cells that once contained a mutated ADE2 gene underwent successful transformation of a wildtype (for ADE2) plasmid DNA. The presence of plasmid DNA in the yeast cells is confirmed by their beige color (Fig. 1). Given the yeast cells were plated on a media that did not contain adenine, it is clear that these cells are in fact making their own adenine and ATP needed to sustain life. Upon transferring approximately 250 phenotypically wildtype, beige cells to a new media containing adenine (Fig. 2), we are able to see 25 cells reverted back to the mutant state, no longer making their own adenine and evidenced by their orange color. In order to revert to a mutant state, the cell must have 1. Temporarily maintained the plasmid independent of chromosomal DNA (never undergoing recombination) and then dropped the wildtype plasmid when placed in a media already containing adenine. Due to the efficiency of bacterial and yeast function; plasmids for unnecessary proteins are typically dropped. The cells containing sectored colors of orange and beige are still considered to have refrained from recombination or including the plasmid genetic material in their chromosomal DNA. It is likely that the orange pigmented precursor has not had time to build up in 100% of the yeast cell giving a sectorial presentation. The 12 beige colored cells have integrated the plasmid DNA into their chromosomal DNA as 100% of the cell shows the pigment of the wildtype ADE gene even when placed on a media containing adenine. While it is unknown how many other, additional genes from the plasmid were integrated into the chromosomal DNA, it is clear that the wildtype ADE gene found on the plasmid are now permanently a part of these cells genome. Given that this chromosomal DNA is passed on to daughter cells during cell division, all of the newly divided cells should also be beige in color.

The process of transformation is one with wide ranging implications. Studies of bacterial and fungal transformation lend necessary insight and discovery of the specific genes that encode proteins used by bacteria to create toxins and antibiotics within the cell. Bacterial cells are constantly sharing plasmids and genetic material by conjugation and transformation, effectively creating new defenses and new strains of cells that are becoming resistant to pharmaceutically made antibiotics. The need to track and identify these genes, extract and study the proteins they code for, as well as understanding the defenses/abilities added to cell by a given gene, allow us to create effective treatments of fungal and bacterial infections. These life-saving medications and treatments are of immeasurable worth and responsible for the preservation of life, and increase in quality of living. For quite some time MRSA, (Methicillin-resistant Staphylococcus aureus), a bacterial infection, was a primary concern to health care workers as this strain of Staph infection had grown resistant to methicillin, the first line treatment of Staphylococcus aureus,. Through bacterial genetics research, anew front-line antibiotic treatment, Vancomycin, was found to be highly effective in eradicating cases of MRSA; however, due to the transfer and shuffling of genetic material through transformation and various

types of conjugation, strains of bacteria like those that cause MRSA are now becoming resistant to Vancomycin, these strains of bacteria are called VRSA (Vancomycin-resistant Staphylococcus aureus). This is just one example of many fundamental and necessary uses of research in bacterial and fungal genetics. Given the highly adept nature of bacteria and fungi, it is unlikely we could ever outrun the evolutions of bacterial and fungal cells, but knowledge and study of the transfer of genetic material in various organisms afford us the opportunity to have a tremendous impact on human lives.

Resources:

Hood, Leroy, Michael L. Goldberg, Ann E. Reynolds, and Lee M. Silver. "Saccharomyces Cerevisiae: Genetic Portrait of a Yeast." Genetics: From Genes to Genomes. By Leland H. Hartwell. 4th ed. Vol. Ref. A. N.p.: n.p., 2011. N. pag. McGraw Hill Higher Ed. Web. <http://highered.mcgraw-hill.com/sites/dl/free/007352526x/387004/Reference_A.pdf>.

Bedwell, David M. "Genetic Model Systems: Yeast." Genetic Model Systems: Yeast. University of Alabama - Birmingham, n.d. Web. <http://www.microbio.uab.edu/bedwell/lectures/notes.pdf>