2011-12 L-113 UV mutagenesis
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2011-12 L-113 UV mutagenesis

Course: BIO-L 113, Spring 2012

School: Indiana

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Mutations and Survival Rate of Yeast exposed to Ultra Violet Radiation Dennis Uzar, Nick Luz L113, Monday, 5:45 PM Section 14987 February 27, 2012 AI: Karen Bohorquez Summary: Using UV irradiation, the effects of UV light on S. cerevisiae were studied and related to all living cells and DNA. Seven plates in a tryptophan deficient medium (SD) were plated with an original undiluted haploid culture and seven plates...

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and Mutations Survival Rate of Yeast exposed to Ultra Violet Radiation Dennis Uzar, Nick Luz L113, Monday, 5:45 PM Section 14987 February 27, 2012 AI: Karen Bohorquez Summary: Using UV irradiation, the effects of UV light on S. cerevisiae were studied and related to all living cells and DNA. Seven plates in a tryptophan deficient medium (SD) were plated with an original undiluted haploid culture and seven plates containing tryptophan (SC) were plated using a 10-4 diluted haploid culture. These 14 agar plates were tested in pairs under a certain allotment of UV exposure. The results showed that percent survival in the SC medium decreased as the time of UV exposure increased and the percent mutations in the SD medium increased as the tome of UV exposure increased. These results occurred because the UV light damaged the DNA of the yeast in the SC medium thus being deadly, while the yeast in the SD medium were beneficially mutated back into the wild type which allowed them to replicate. This experiment supports the hypothesis that UV light can damage and mutate DNA in living cells. Methods: An Eppendorf tube containing an overnight culture of Saccharomyces cerevisiae (yeast) that had the trp1-289 allele was created by incubating the culture overnight at 30 C and supplied for the experiment as the original haploid culture. Four other Eppendorf tubes were labeled 10-1, 10-2, 10-3, and 10-4 and filled with 900 l of H2O to prepare for the serial dilutions. For the tube labeled 10-1, 100 l of the original haploid culture was pipetted and mixed thoroughly to create the first dilution. For the proceeding dilutions, pipet tips were changed each time and 100 l from the 10-1 tube was pipetted into the 10-2 tube. The suspension from the 10-2 tube was mixed and 100 l was pipetted into the 10-3 tube and mixed again. Lastly, 100 l of solution from the 10-3 tube was pipetted into the 10-4 tube to create the last serial dilution. After the serial dilutions were completed, seven petri dishes containing a synthetic complete medium (SC) and seven petri dishes containing a synthetic dropout medium (SD-trp-), which lacked tryptophan, were acquired and labeled accordingly. The seven SC petri dishes all received 100 l of the 10-4 tube each and were promptly covered back up to avoid any other bacteria landing on the agar and growing. Once each petri dish had 100 l of 10-4 solution a glass spreader was used to evenly spread the yeast in the agar. To avoid any contamination the spreader was disinfected by placing it in 95% alcohol and passing it through a flame to burn off the residual alcohol. The same procedure was used for the seven plates labeled SD-trp- except, 100 l of the original haploid culture was added to each instead of the diluted culture (Bonner et. al. 2012). The yeast cells were then tested under certain durations of UV light. A control was created by setting aside one plate from each the SC medium and SD medium and labeled as 0 seconds of exposure. Exposure to UV radiation was 20, 40, 60, 80, 100, and 120 seconds from a short wave UV lamp that provides UV radiation at approximately 250nm. A piece of aluminum foil covered the UV lamp allowing exposure time to be more accurate because it was difficult to turn the lamps on and off with precision. One set of plates (one SD and one SC) were labeled 20 seconds and placed equal distances below the lamp, their lids removed so the UV rays can reach the cells and a sheet of saran wrap was promptly placed on top of both plates to prevent contamination. The sheet of aluminum foil was removed from the UV lamp and the set of plates received exactly 20 seconds of exposure before the foil was replaced over the UV lamp. The saran wrap removed, was then the lids placed back on and the plates set aside. A template was made by tracing the location of the first set of plates onto a piece of paper taped under the UV lamp to ensure exact placement for the rest of the plates. This process was repeated for the 40, 60, 80, 100, and 120 second pairs, increasing the time of UV irradiation appropriately for each set of pairs. Once completed we had obtained seven SC medium plates with increasing UV exposure time and seven SD-trp- medium plates with increasing UV exposure time. The 14 plates were stacked upside down and incubated for two days at 30 C then refrigerated until the next lab to prevent any more growth. One week later the number of colonies on each plate was recorded and the data was compiled as a class. Using the class data (from SC plates), percent survival of the colonies according to duration of UV exposure was calculated as follows: The zero second plate is the control because it received no UV exposure so it was assumed that 100% of the cells survived. Mutation rate was also calculated using the class data from the same duration of UV exposure as follows: Once the percent survival and percent mutation for each time interval of UV irradiation were calculated, MS Excel was used to plot graphs of % survival vs. time of UV exposure and % mutation vs. time of UV exposure. Results: The percent mutation rate of the SD plated yeast increased with longer UV exposure, but eventually leveled out at about .006% mutation (Figure 1). This shows that to a certain degree increase in UV radiation can increase mutation but eventually there is a point where more UV radiation will not do anything. The percent survival rate of the SC plated yeast decreased with increases in time of UV exposure, but again leveled off at about 5% survival (Figure 2). This also shows that to a certain degree, increases in UV radiation decrease percent survival but only to a certain degree. Figure 1 Effect of time of UV exposure on the percentage of mutation rates of yeast Figure 2 Effect of time of UV exposure on the percentage of survival rate of yeast. Discussion: The effect UV irradiation on the overall survival of yeast cells was that it damaged the DNA of the yeast cells because the percent survival of the yeast cells continued to decline as the time of UV exposure increased. The UV induced genetic changes in the trp1-289 cells because they were lacking tryptophan and the mutations caused the yeast cells to mutate back into fully functional cells that produce tryptophan (Tomoyuki et al. 2012). We know this is true because if no mutations occurred that caused the cells to revert back into the wild type, there would be no yeast growth because of the lack of tryptophan. According to the data it can be argued that UV kills yeast and that DNA is damaged because percent survival of the tryptophan producing yeast declined as UV exposure increased and the percent mutation of the trp1-289 increased as UV exposure increased. The curve for the reversion of trp1-289 shows the shape that it does because to a certain extent more UV exposure beneficially mutates the yeast but above a certain level of exposure to UV light there is no more increase in mutation and thus no more benefit. Refrences Bonner, J. Hengeveld, J. Holdeman, R. Ruf, W. Rynkiewicz, E. 2012. Basic Physiology and Biochemistry of Chloroplast Membranes. Biology L113 Laboratory Manual, Spring 2012. Plymouth, MI: Hayden-Mcneil Publishing. Pp C1-C10. Tomoyuki Fujii, et al. "Thermosensitivity Of A Barosensitive Saccharomyces Cerevisiae Mutant Obtained By UV Mutagenesis." High Pressure Research 30.4 (2010): 524-529. Academic Search Premier. Web. 27 Feb. 2012.

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