Cori DeCicco Experimental Biology Yeast Model Introduction Yeast is an important experimental tool used in biology.

Yeast is easy to grow with a quick doubling time and it also possesses genes similar to disease causing genes in humans. In this experiment, yeast was used as a model system to study DNA damage. To cause DNA damage, a drug called hydroxyurea (HU) was used. HU is known to stall the replication fork of yeast. Because of this stalling, DNA synthesis is not able to happen. When damage happens in a normal, healthy yeast cell, a signal is sent to sensor proteins which then tell response protein which will send for DNA repair. Mutations can cause this pathway to not function properly. If the sensor protein is not working properly, then DNA damage could go unnoticed by the cell until after replication. If there was a mutation in the response protein, then DNA damage would be sensed and replication could be stopped but there would be nothing to promote repair. The protein pathway is an important part of making sure a cell is not damaged and is functioning properly. In this experiment, the following strains were used: wild type (WT), MutX, MutY, MutXY. WT did not have any genes knockout out and it was known that Mut XY had both sensory and response proteins knocked out. MutX and MutY either had a senor protein or a response protein knocked out. Which strain had each mutation was unknown. By using techniques in the lab, such as examining the effects of HU on yeast cultures, extracting RNA from yeast, making these cultures into cDNA for PCR, and analyzing PCR products by gel electrophoresis, this experiment tried to find out which strain had each of the mutations.

Centrifuge for 15 sec at 8000x g. Discard flow-through. Add 10uL of DNase I stock solution to 70uL Buffer RDD. Add 80uL of DNase I incubation mix to spin column membrane and place on benchtop for 15 minutes. 25mL of yeast culture was added with a concentration of 200mM of HU. Transfer about 700 uL into RNeasy spin column in a 2mL collection tube. Mix gently and centrifuge briefly to collect residue from sides of tube. Yeast cultures were streaked on YPD media in presence or absence of HU. The frozen cells were then incubated in a water bath at 37C for about 5 minutes until thawed. In the +HU tubes. Two petri dishes: 1 was YPD while the other was YPD+HU were divided into 4 quadrants for each of the yeast strains: WT. Each culture contained about 5x 10^7 cells. 350mL of 70% ethanol was then added to cell lysate and mixed by pipetting. The cultures were then incubated. 1mL of each strain was plated with a wire loop onto each of the petri dishes. Liquid cultures of each yeast strain were prepared in presence or absence of HU. 3. Add 350uL Buffer RW1 to spin column and centrifuge . Lysates were prepared by letting each yeast strain grow until OD600~0. Reuse collection tube. Cells were then frozen and stored at -80C. The yeast cultures and media were mixed so that 50mL of culture so that OD600 = 0. After one hour. Discard flowthrough and reuse collection tube. The strains were split 200mM of HU was added to one of the cultures of each strain.Material and Methods 1.15. MutXY.3 at room temperature. Add 350uL of Buffer Rw1 to RNeasy spin column. Each culture was put into the spectrometer and the absorbance was recorded every 20 minutes. 2.MutY. Centrifuge for 15sec at 8000 x g. the cells were collected and resuspended in RLT buffer and lysed for 3 minutes on a mini-bead beater. MutX.

HUG1-forward. Add 1uL of DEPC-treated water to tube 3. 3uL of DEPC-treated water. spin sample briefly. Add 1uL taq polymerase to each reaction and pipette to . In tube 3 add 5uL of +HU total RNA.5mL) and add 30uL RNase free water to spin column membrane. Add the following to PCR tubes. Label 3 of the 8 PCR tubes with #s 1. Centrifuge at full speed for 1 min. Add the follow to each tube: 2. Incubate at 42C for 2 minutes. In tube 1 add 5uL of –HU total RNA.3 and which yeast strain. Once thawed. 5uL 10X taq buffer. Add 1uL SuperScript II to tubes 1 and 2. add 1uL RNase H enzyme and incubate for 20 minutes at 37C. To rxn #2 add 3uL of +HU. Incubate at 65C for 5 minutes and then place on ice for 1 min. Briefly spin down mixtures.-RT cDNA sample. Place spin column in new collection tube (1. Add 500uL of Buffer RPE to spin column and centrifuge for 15 sec at 8000 x g. 1uL 10mM dNTP mix.5uL of each. Add the following to each mixture: 2uL 10x RT. In tube 2 add 5uL of +HU total RNA.2. Thaw sample collected in step 3 on ice. Add another 30uL of RNase free water and centrifudge at full speed again for 1 min. 1uL RNaseOUT. not sample tubes: 30uL sterile water.1M DTT.2. ACT1-forward. 5. Act1-Reverse. Discard flow through and add 500uL Buffe RPE to spin column and centrifuge for 2 minutes at 8000 x g. 4.-RT cDNA sample. To rxn#3 add +HU. 2uL 0. 4uL 25mM MgCl2. Label 3 PCR tubes 1. +RT cDNA sample to rxn #1. Discard flow through and follow first Buffer RPE wash step. HUG1-reverse. Put tubes in thermal cycler and run RT program. Thaw samples collected from step 4. Store at -80C. The final volumne in the collection tube should be 60uL of RNA. 1uL of primer. Add 3uL of –HU.for 15 sec at 8000 x g. Add each of the following to each tube: 1uL of 10mM dNTP mix.3 and yeast strain. Place spin column in new collection tube and centrifuge at full speed for 1 min.

Run at ~120V in 1X TAE buffer.mix. Load ~25uL of each sample in 1. Results Figure 1 . Soak gel in SybR green for 30 minutes and take picture of gel.5% agarose gel wells along with DNA ladder. Briefly spin down rxns and put tubes in thermal cycler and run the proper program.

MutY. .Figure 1 shows yeast cultures of the following strains: WT. MutX. Each of these yeast strains were grown on both YPD and YPD+HU media. MutXY.

recording the absorbance once every 20 minutes. 50mL of each culture of the yeast strain was prepared at OD600 = 0.8 0.2 0.6 0.Figure 2 Doubling Times of Yeast Strains WT.4 0.7 Absorbance OD600 0.1 0 0 50 100 150 200 250 300 Time (minutes) WT -HU mutY -HU WT +HU mutY +HU mutX -HU mutXY -HU mutX +HU mutXY +HU Figure 2 is depicting the growth rate and doubling times of each strain of yeast in presence or absence of HU. MutY.15. . The absobance of the cultures were observed for 310 minutes.5 0. MutX. Mut XY in presence and absence of HU 0.3 0.

001 x+0.0005 x+0.4 1386 Table 1 shows the rate of growth and doubling times for each of the yeast strains seen in Figure 2.000 8x+0.000 7x+0.168 6 mutXY +HU y=0.1784 533.4 630.1 990.21 18 mutY +HU y=0.20 57 mutX +HU y=0. The rate of growth is equal to the slope of the line.001 3x+0.21 11 mutXY -HU y=0.20 37 WT +HU y=0.23 45 mutY -HU y=0.2 693.2 990.Table 1 gro wth rate dou blin g tim e WT-HU y=0.001 1x+0.2 495.22 68 MutX -HU y=0. The double time was calculated using the equation DT=(ln(2))/r where r is the rate of growth (slope) .1 866.000 7x+0.001 4x+0.

This data was collected initially and at the end time (310 minutes).Figure 3 % of Budding at 0 minutes and 310 minutes 100 80 60 40 20 0 WT -HU WT +HU mutX -HU mutX +HU mutY -HU mutY +HU mutXY HU mutXY +HU % budded % budded at 0 min % budded at 310 min Figure 3 is a bar graph displaying the percent of budding of each strain of yeast seen in Figure 2 in liquid culture in the presence or absence of HU. .

and Column 4 had XY/+HU.5% agarose gel which was the gel electrophoresis was ran at ~120V.Figure 4 Figure 4 shows stained gel from gel electrophoresis. Column 1 had the DNA ladder loaded. -RT. The gel was then stained with SyBr green. The gel contained yeast strain XY in the presence or absence of HU but also in the presence or absence of reverse transcriptase. The gel was a 1. Column 3 had XY/+HU. The other columns were not our samples and will not be discussed here. . Column 2 had XY/-HU. +RT. +RT. Each sample was mixed with loading dye.

As seen in Table 1. Figure 2 and 3 along with Table 1 were all obtained after running liquid cultures of each yeast strain through a spectrometer. it would be expected to have the same outcome as a mutation solely in the sensor protein. shows that the % budded for MutX in the presence of HU stayed relatively consistent with MutX in the absence of HU. As for MutY. the WT strain’s doubling time was greatly extended in the presence of HU.Discussion Figure 1 shows each of the yeast strains grown on YPD and YPD+HU. This shows some evidence for MutY having a mutation in a response protein because the strain would still have the means of initiating cell arrest which would slow down growth rate. one reason why it grew was that it was missing sensor protein because it would not stop replication in the presence of HU. Since the sensor protein is more upstream in the pathway than the repair protein. only WT and MutY grew on this media. What is strange is that MutXY did not have any growth MutXY has a double knockout. which does not tell much but looking at Figure 3. then replication could continue. like WT. YPD+HU has hydroxyurea which causes DNA damage in yeast. missing both the sensor and response/repair protein. Looking at figure 3 also supports this hypothesis because the percentage of budded cells in presence of HU at 310 minutes is close to 100%. The doubling time for MutX was also increased. This would make sense for a cell with a mutation in the . This is because the WT strain has the capability to initiate cell cycle arrest in the presence of damage (HU) but will keep replicating once the damage is repaired. As seen in Figure 1. It would make sense why WT would grow because if they cells were able to repair themselves. YPD is normal yeast media which all strains can grow on. The doubling time of MutY in the presence of HU shown in Figure 2 is the same as WT in presence of HU.

it can be seen that MutY+HU looks very similar to WT+HU. Using the data. Looking at Figure 2 and Table 1. The MutX strain may have a been a mutation in the sensory protein. RNase is found everywhere and could have gotten into the sample and degraded it. There are a few errors that could have happened. the gel electrophoresis signals were not very strong which could mean more copies of the cDNA could have been made with PCR to give a stronger signal in the gel. This means a few different things could have happened. Figure 4 shows the stained aragose gel with yeast strains XY in presence and absence. it can also be assumed that MutY has the response protein mutation. If MutX did have a mutation in the sensory protein. There seems to be no PCR product. it would be expected that the growth rate in response to HU would not be affected since the cell lacks the protein to sense DNA damage. By looking at the data obtained from this experiment. there could have been an issue with obtaining the total RNA. This may mean that there was no mRNA and no cDNA was transcribed. Evidence for this is shown in Figure 2 where the DT for MutX+HU is larger than –HU but not as large as the other strains. This shows that the cells would still be going through the cell cycle without detecting DNA damage. First. Figure 3 would also be consistent with this assumption since the budding % stayed the same in MutX+HU at 0 minutes and at 310 minutes. One explanation for this could be that fact that the cells with much DNA damage would end up dying and thus altering the growth rate and doubling time. This shows that MutY asks as WT would in presence of HU because . Also. If no cDNA was transcribe this means there was nothing for PCR to amplify.response protein because these cells can still initiate cell arrest and thus cell cycle would be stopped while the cells were budded. an educated guess about which strain has which mutation can be made. This is not what was observed in the data in Figure 2 and Table 1.

the cell still has the protein to initiate cell arrest. By studying this model. . it cannot sense DNA damage in the cell and will keep replicating with this damage. By understand how damage affects protein pathways in yeast cultures. Looking at DNA damage in yeast is an important model in biology. If a cell is mutated at the sensory protein. One way to relate this model to humans is thinking about cancer. Figure 3 also shows evidence for MutY have a response protein mutation since most of its cells at 310min at budded which shows that the cell cycle was stopped. The yeast model also serves as a good tool for testing drugs that may affect these pathways. Although the cell sensed that DNA damaged had occurred. it does not possess the mechanism to fix the damage. research may progress to find a cure or treatment for cancer and other disease similar that affect the protein pathway. we can begin to understand how this type of damage may affect humans.

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